Optimization of gene sequences of chimeric virus-like particles for expression in insect cells

ABSTRACT

Chimeric virus-like particles that exhibit conformational antigenic epitopes capable of eliciting neutralizing antibodies are disclosed herein. The chimeric virus-like particles of the invention comprise a recombinant viral capsid protein that encapsulates a recombinant viral protein during self-assembly into a chimeric virus-like particle, wherein the chimeric virus-like particle exhibits confirmational antigenic epitopes capable of eliciting neutralizing antibodies. Pharmaceutical compositions, vaccines, and diagnostic test kits containing the chimeric virus-particles are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 37 U.S.C. §119(e) based on U.S.Provisional Application Nos. 60/356,119, 60/356,161, 60/356,118,60/356,133, 60/356,157, 60/356,156, 60/356,123, 60/356,113, 60/356,154,60/356,135, 60/356,126, 60/356,162, 60/356,150, 60/356,151, and60/356,152, each filed Feb. 14, 2002, the entire contents of each ofwhich are incorporated herein by reference.

I. FIELD OF THE INVENTION

The present invention relates to the field of viral vaccines,therapeutics, and diagnostics, compositions and methods for thedetection, protection and treatment of human papillomavirus (HPV)infections and associated dysplasia. In particular, the inventionrelates to novel polynucleotide molecules encoding recombinant HPV geneproducts having increased antigenicity and immunogenicy in mammals.

II. BACKGROUND OF THE INVENTION

Cervical cancer results in over 200,000 deaths per year worldwide(Paildn et al., 1990; Pisani et al., 1990). The greatest burden ofdisease is in developing countries, where cervical cancer is the mostfrequent female malignancy and comprises 25% of all female cancers.Cervical dysplasia makes up 7% of all female cancers and causes greaterthan 5000 deaths per year in the U.S. (Shah and Howley, 1996). Throughclinical studies, epidemiologists have identified human papillomavirus(HPV) as the major cause of cervical cancer and cervical dysplasia.(Walboomers et al., 1999). On a worldwide basis, most cervical cancerscontain the genes of “high-risk” HPVs (genotypes 16, 18, 31, and 45)(Bosch et al., 1995; Walboomers et al., 1999). The nucleotide sequencesof human and animal papillomavirus genomes are accessible in GenBank.

HPV-16 is found in approximately 50% of cervical cancers, and HPV-18,HPV-31, and HPV-45 account for an additional 25-30% of HPV-positivetumors. Though early detection of HPV-induced cervical neoplasia ispossible with Papanicolau (PAP) smears and cervicoscopy, screeningprograms in developing countries are only now emerging. In the UnitedStates, where the widespread availability of PAP screening and othermethods have been associated with a reduction in the incidence ofcervical cancer, the annual economic loss in the U.S. is still estimatedat $5 billion (Kimbauer et al., 1993). Effective HPV vaccines wouldreduce the prevalence of worldwide cervical cancer and reduce the costof screening and treating premalignant cervical disease.

Prophylactic viral vaccines that efficiently prevent infection or modifydisease have a successful record as cost-effective approaches to preventand manage viral diseases. Human papillomaviruses are DNA tumor virusesthat encode several viral oncogenes. Two of these viral oncogenes, E6and E7, are conserved and expressed in human genital warts, dysplasia,and tumors, and may be required for maintenance of the tumorigenicphenotype. These features raise theoretical arguments against a HPV E6and/or E7 subunit protein or DNA vaccine consisting of these viralproteins alone. Wild type and intact versions of these viral genes andtheir gene products in the context of a vaccine may disrupt normal hostcell gene regulation by increasing the levels of Rb and p53 proteins andfacilitate cell transformation. Subunit protein viral vaccines utilizingvirus-like particles (VLPs), analogous to the hepatitis B virus vaccinederived from yeast, have been developed as prophylactic vaccines toprevent viral infections and diseases including HPV infections (Schillerand Lowy, 1996; Cook et al., 1999; Harro et al., 2001).

Papillomaviruses encode the major capsid gene, L1, whose gene productsare able to self-assemble into virus-like particles in the absence ofother viral gene products (Kirnbauer et al., 1992; Hagensee andGalloway, 1993; Kirnbauer et al., 1993). Recombinant papillomavirus L1VLPs display several properties that are advantageous for vaccines.These features include the following: (1) similar size and morphology asnatural papillomavirus virions as shown by electron microscopy, (2)immunodominant and conformational epitopes present on natural virions asdetermined by immunodetection assays with neutralizing monoclonalantibodies, and (3) elicitation of high titers of type-specificneutralizing antibodies as seen in sera of vaccines (Kirnbauer et al.,1992; 1993).

Several trials of preventive papillomavirus vaccine candidates using L1VLPs purified from insect cells have been conducted in animals using thecutaneous cottontail rabbit papillomavirus (CRPV) disease model, theoral mucosal bovine papillomavirus 4 (BPV4), and canine oralpapillomavirus (COPV) models in cattle and dogs, respectively. Threesubcutaneous injections of CRPV L1 VLPs given without adjuvant, orcombined with alum or Freund's adjuvant, protected rabbits for at leastone year against persistent infection and subsequent carcinoma afterhigh-dose CRPV challenge (Breitburd et al., 1993; Christensen et al.,1996). Similarly, calves and dogs given two intramuscular injections ofBPV4 L1 VLPs (with alum) and COPV L1 VLPs (without adjuvant),respectively, were protected from subsequent oral mucosal challenge(Suzich et al., 1995; Kirnbauer et al., 1996). In the CRPV and COPVmodels, passive transfer of serum or IgG from animals immunized with theL1 VLPs protected animals challenged with the homologous virus,indicating that neutralizing antibodies were sufficient to conferprotection (Breitburd et al., 1993; Suzich et al., 1995).

Recombinant peptides or proteins encoded and expressed by customsynthesized genes often require further modifications. These peptideshave often lost their ability to fold and show no disulfide bondformation. Thus proteins frequently are not stable in the presence ofendogenous bacterial proteases, and tend to aggregate into inactivecomplexes. Consequently, recombinant peptides often suffer from lowyield and demonstrate reduced antigencity and immunogencity as comparedwith native peptides.

Purification of heterologous recombinant proteins frombaculovirus-infected insect cells demonstrated that host contaminantproteins were best separated from the recombinant protein using an ionexchange step as the first step in the protocol (Robinson et al., 1998).Purification of baculovirus-derived HPV L1 VLPs (Kirnbauer et al, 1993;Suzich et al., 1995) and yeast-derived intracellular HPV L1 VLPs (Cooket al., 1999) were described previously.

The invention as disclosed and described herein, overcomes the prior artproblems with HPV therapies through the generation of novel syntheticpolynucleotides that encode HPV capsid genes encoding HPV capsidproteins capable of assembly into VLPs. The capsid proteins of theinvention retain their optimum native folding and exhibit conformationalpresentation of epitopes that elicit antigen-neutralizing antibodies.Large scale production and purification of HPV-L1 VLPs and HPV chimericVLPs and their manufacturing for vaccines and other pharmaceuticalproducts are also disclosed.

III. SUMMARY OF THE INVENTION

This invention is directed toward the prevention, treatment, anddiagnosis of papillomavirus infections and associated benign andneoplastic diseases in humans. In particular, the invention disclosesnovel synthetic polynucleotides capable of expressing highly immunogenicHPV VLP and HPV chimeric VLP products.

According to one aspect of the invention, there is provided a chimericvirus-like particle comprising a recombinant viral capsid protein thatencapsulates a recombinant viral protein during self assembly into achimeric virus-like particle, wherein the chimeric virus-like particleexhibits conformational antigenic epitopes capable of elicitingneutralizing antibodies the recombinant viral capsid protein and therecombinant viral protein are from the same or a different virus.

In one embodiment, the invention provides a chimeric virus-like particleof a human papillomavirus. Preferably, the human papillomaviruscomprises genotypes HPV-16, HPV-18, HPV-45, BPV-31, HPV-33, HPV-35,HPV-5 1, HPV-52, HPV-6, HPV-11, HPV42, HPV-43, HPV-44, or a combinationthereof. In another embodiment, the invention provides a chimericvirus-like particle wherein the viral gene is a L2 fusion gene. The L2gene is fused with other papillomavirus genes or other viral genes toform a heterologous gene.

In another embodiment, the viral capsid protein, the viral protein, orboth are encoded by codon-modified polynucleotides comprising SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5, or apolynucleotide having a sequence that is substantially homologous tothese SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, or SEQ IDNo. 5.

In another aspect, the invention provides pharmaceutical compositionsfor treating, ameliorating, or preventing a papillomavirus relateddisease or disorder comprising a multiplicity of chimeric virus-likeparticles of the invention that elicite conformational antigenicepitopes, and an acceptable carrier or diluent.

In one embodiment, the pharmaceutical composition comprises: (a) apolypeptide which is encoded by a polynucleotide molecule comprising SEQID No., or a polynucleotide having a sequence that is substantiallyhomologous to SEQ ID No. 1, (b) a polynucleotide molecule-comprising SEQID No. 1, or a polynucleotide having a sequence that is substantiallyhomologous to SEQ ID No. 1; (c) or a vector carrying a polynucloetide amolecule comprising SEQ ID No. 1, or a polynucleotide having a sequencethat is substantially homologous to SEQ ID No. 1, and a pharmaceuticallyacceptable carrier or diluent.

In yet another aspect, the invention provides a vaccine composition toinduce immunity against a papillomavirus infection in human comprisingadministering to a subject a composition containing chimeric virus-likeparticles that exhibit conformational antigenic epitopes capable ofeliciting neutralizing antibodies in a subject, wherein the chimericvirus-like particles comprise a recombinant HPV L1 that encapsulates arecombinant HPV L2 and/or HPV L2 fusion protein, and a detection agent.The vaccine provides humoral immunity, cell-mediated immunity, or both.The vaccine protects against papillomavirus infections that are causedby one or more human papillomaviurs genotypes.

In another aspect, the invention provides a diagnostic test kit fordetection of papillomavirus infection comprising a compositioncontaining chimeric virus-like particles that exhibit conformationalantigenic epitopes capable of eliciting neutralizing antibodies and adetectable agent.

In yet another aspect, the invention provides a method for preparing acodon-optimized polynucleotide comprising one or more of the followingsteps: (a) replacing codons that are underutilized in insect cells withcodons that are utilized at high levels in insect cells, to create aninitially-modified nucleotide sequence; (b) modifying theinitially-modified nucleotide sequence by choosing a preferred codon forthe initially modified sequence, wherein: (i) the ratio of GC nucleotidepairs to AT nucleotide pairs in the further-modified nucleotide sequencetrends toward approximately 1:1; (ii) the number of palindromic andstem-loop DNA structures in the further-modified nucleotide sequence isminimized; and (iii) the number of transcription and post-transcriptionrepressor elements are minimized.

In another aspect, the invention provides a method of treating,ameliorating, or preventing a papillomavirus-related disease or disordercomprising administering to an individual in need thereof an effectiveamount of the pharmaceutical composition of the invention.

IV. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show the alignment of two wild-type HPV-16 papilloma virusL1 polynucleotide sequences with a codon-optimized HPV-16 L1polynucleotide of the invention. The aligned sequences are: HPV-16 L1wild-type sequence from GenBank record Accession No.

K0278 (“11gbseq”; SEQ ID No. 12), HPV-16 wild type clone NVAX (“11nvax”;SEQ ID No. 11), and HPV-16 codon-optimized L1 (“11optmzd”; SEQ ID No.1).The sequences were aligned using the Gene Runner™ program (HastingsSoftware) available through the website maintained by the NationalCenter for Biotechnology Information (NCBI). Nucleotides which differbetween the aligned sequences are boxed.

FIG. 2 shows a schematic flowchart of the steps in the weaning selectionprocess of an insect cell line capable of growing in serum-free media assuspension cell cultures.

FIG. 3 shows a schematic flowchart of the steps in the protein secretionselection process of an insect cell line capable of growing inserum-free media as suspension cell cultures and of enhanced expressionof extracellular recombinant proteins and virus-like particles.

FIG. 4 shows a photomicrograph of a confluent monolayer of Sf-9S insectcells grown in serum-free insect cell media (Sf-900 II SFM, GIBCO)visualized by inverted phase-contrast microscopy at 400× magnificationusing Kodachrome 100 color film (Kodak).

FIG. 5 shows a schematic flowchart of the basic steps in the productionor manufacturing of purified HPV VLP products.

FIG. 6 shows a schematic flowchart of the steps in upstream processingof baculovirus infected insect cell suspensions for production ofrecombinant HPV L1 VLPs.

FIG. 7 shows a schematic flowchart of the steps in upstream processingof baculovirus-infected insect cell suspensions for production ofrecombinant HPV chimeric VLPs.

FIG. 8A shows a schematic flowchart of the downstream processing methodfor purification of recombinant HPV VLPs by continuous flowultracentrifugation using linear sucrose gradients.

FIG. 8B shows a schematic flowchart of the downstream processing methodfor purification of recombinant HPV VLPs by column chromatography usingion exchange and affinity binding matrices.

FIG. 8C shows a schematic flowchart of the downstream processing methodfor purification of recombinant HPV VLPs by ultracentrifugation usingdiscontinuous sucrose step gradients.

FIG. 9 shows a stained protein gel of the products of the invention(i.e., baculovirus-derived recombinant HPV-16 L1 VLPs purified accordingto the methods of the present invention.

FIG. 10A shows proteins detected chromogenically on membranes by Westernblot analysis of recombinant HPV-16 L1 VLPs purified according to themethods of the present invention and bound to polyclonal antisera toHPV-16 L1 protein (1:10,000).

FIG. 10B shows proteins detected chromogenically on membranes by Westernblot analysis of recombinant HPV-16 L1 VLPs purified according to themethods of the present invention and bound to polyclonal antisera toSf-9S insect cell proteins (1:500).

FIG. 10C shows proteins detected chromogenically on membranes by Westernblot analysis of recombinant HPV-16 L1 VLPs purified according to themethods of the present invention and bound to polyclonal antisera toAcMNPV wild-type baculovirus (1:500).

FIG. 11 shows a graph of the binding results of H16.V5 murine monoclonalantibody to conformational epitopes on untreated and Triton X-100-treated recombinant HPV-16 L1 VLPs purified and treated according tothe methods of the present invention as measured by enzyme linkedimmunoadsorbent assay (ELISA) analysis.

FIG. 12 shows a chromatogram of a product of the invention, recombinantHPV-16 L1 VLPs purified according to the methods of the presentinvention, analyzed by analytical size exclusion chromatography.

FIG. 13 shows an electron micrograph of baculovirus-derived recombinantHPV-16 L1 VLPs purified according to the methods of the presentinvention, stained negatively with uranyl acetate, and magnified36,000×. The bar scale is 50 nm.

V. DETAILED DESCRIPTION OF THE INVENTION

The invention, as disclosed and described herein, provides compositionsand methods for detecting, preventing, ameliorating, or treatingpapillomavirus related diseases and disorders. The pharmaceuticalcomposition of the invention contains recombinant viral proteins thatself assemble into virus-like particles (VLPs) exhibiting conformationalantigenic epitopes capable of raising neutralizing antibodies. The VLPsof the invention are expressed from a host cell extracellulary,intracellularly, or both.

Definitions

The definitions used in this application are for illustrative purposesand do not limit the scope of the invention.

As used in herein, “virus-like particles” or “VLPS” refers to virusparticles that self-assemble into intact virus structures comprised ofcapsid proteins such as papillomavirus L1 capsid proteins. VLPs aremorphologically and antigenically similar to authentic virions, but donot contain genetic information sufficient to replicate and thus arenon-infectious. VLPs are produced in suitable insect host cells (i.e.,yeast, mamalian, and insect host cells), wherein upon isolation andfurther purification under suitable conditions, are purified as intactVLPs.

As used herein, “chimeric VLP” refers to recombinant papillomavirus L1capsid protein, or peptide fragment thereof, that encapsulates otherpapillomavirus gene products or heterologous gene products duringself-assembly into virus-like particles. For example, gene productscontaining the HPV L2, E2, E6, and/or E7 and which become encapsulatedinto the HPV L1 VLPs are considered herein as chimeric VLPs.

As used herein, “L2 fusion protein” refers to a protein, or a peptidefragment thereof, encoded by a papillomavirus L2 scaffolding gene fusedto papillomavirus or other viral genes including heterologous gene(s).

As used herein, “heterologous viral capsid genes” refers to viral genesencoding the major structural virion component from different viruses,for example, the rotavirus VP2, VP6, HPV-16 L2, and HPV-16 L1 genes.

As used herein, “protein” is used interchangeably with polypeptide,peptide and peptide fragments.

As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrid,anti-sense RNA, ribozyme, genomic DNA, synthetic forms, and mixedpolymers, both sense and antisense strands, and may be chemically orbiochemically modified to contain non-natural or derivatized, synthetic,or semi-synthetic nucleotide bases. Also, included within the scope ofthe invention are alterations of a wild type or synthetic gene,including but not limited to deletion, insertion, substitution of one ormore nucleotides, or fusion to other polynucleotide sequences, providedthat such changes in the primary sequence of the gene do not alter theexpressed peptide ability to elicit protective immunity.

As used herein, “gene products” include any product that is produced inthe course of the transcription, reverse-transcription, polymerization,translation, post-translation and/or expression of a nucleotidemolecule. Gene products include, but are not limited to, proteins,polypeptides, peptides, or peptide fragments.

As used herein, “L1 protein” refers to the structural protein ofpapillomavirus L1 capsid genes and constitutes the major portion of thepapillomavirus (“PV”) capsid structure. This protein has reportedapplication in the preparation of HPV vaccines and diagnostic reagents.

As used herein, “L2 protein” refers to the structural scaffoldingprotein of papillomavirus, which constitutes a minor portion of thepapillomavirus capsid structure and facilitates the assembly ofpapillomavirus particles within cell nuclei.

As used herein,“L2/E7 protein” refers to a fusion protein, or a fragmentthereof, encoded by a papillomavirus L2 scaffolding gene fused to apapillomavirus E7 transforming gene that may have one or more mutations.

As used herein, “L2/E7/E2 protein” refers to a fusion protein, or afragment thereof, encoded by a papillomavirus L2 scaffolding gene fusedto (a) papillomavirus E2 transactivation gene that may have mutationsand (b) a papillomavirus E7 transforming gene. The fused gene includesone or more mutated genes.

As used herein, “L2/E6 protein” refers to a fusion protein, or a peptidefragment thereof, encoded by a papillomavirus L2 scaffolding gene fusedto a papillomavirus E6 transforming gene that may have one or moremutations.

As disclosed herein, “mutation” includes substitutions, transversions,transitions, transpositions, reversions, deletions, insertions, or otherevents that may have improved desired activity, or a decreasedundesirable activity of the gene. Mutation encompasses null mutations innatural virus isolates or in synthesized genes that may change theprimary amino acid sequences of the expressed protein but do not affectthe self-assembly of capsid proteins, and antigenicity or immunogenicityof VLPs or chimeric VLPs.

As disclosed herein, “substantially homologous sequences” include thosesequences which have at least about 50%, homology, preferably at leastabout 60-70 %, more preferably at least about 70-80% homology, and mostpreferably at least about 95% or more homology to the codon optimizedpolynucleotides of the invention.

As used herein “vaccine” refers to compositions that result in bothactive and passive immunizations. Both polynucleotides and theirexpressed gene products are used as vaccines.

As used herein “biologically active fragments” refer to fragmentsexhibiting activity similar, but not necessarily identical, to anactivity of the viral polypeptide of the present invention. Thebiologically active fragments may have improved desirable activity, or adecreased undesirable activity.

As used herein “polypeptides” include any peptide or protein comprisingtwo or more amino acids joined to each other by peptide bonds. As usedherein, the term refers to both short chains, which also commonly arereferred to in the art as peptides, oligopeptides and oligomers, forexample, and to longer chains, which generally are referred to in theart as proteins, of which there are many types. “Polypeptides” include,for example, biologically active fragments, substantially homologouspolypeptides, oligopeptide, homodimers, heterodimers, variants of thepolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, agonists, antagonists, or antibody of the polypeptide, amongothers. The polypeptides of the invention are natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The terms “amino acid” or “amino acid sequence,” as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments,” “immunogenic fragments,” or“antigenic fragments” refer to fragments of viral proteins which arepreferably at least 5 to about 15 amino acids or more in length, andwhich retain some biological activity or immunological activity of theviral protein.

As used herein, “purity” refers to the amount of intact VLPs present ina final product of the invention.

As used herein, “yield” refers to the amount of purified intact VLPs asa function of the wet weight or the number of initial cells infectedwith recombinant baculoviruses expressing VLPs. For example, a preferredyield of intact VLPs is greater than about 10 mg of VLPs per 10⁹ hostcells.

As used herein, “antigenic characteristic(s)” refers to the ability ofHPV VLPs to bind or cross-react with antisera generated againstwild-type HPV virions of the 'same genotype. Antisera generated byimmunization of animals or humans with HPV VLPs produced according tothe present invention contains immunoglobulin molecules that sharebinding sites of native HPV virions with antisera from humans infectedwith HPV of the same genotype.

1. Sf-9S Cell Line

According to one aspect of the invention described herein, there isprovided a novel cell line designated as Sf-9S, which was deposited ascell line ATCC PTA-4047 on Feb. 4, 2002, under the Budapest Treaty, withthe American Type Culture Collection (ATCC), located at 10801 UniversityBoulevard, Manassas, Va. 20110. The Sf-9S cell line is derived from theparent S. frugiperda Sf-9 cell line (ATCC CRL-1771) and established byclonal selection based on serum-independent growth. This cell line isused as a host cell substrate in a single cell suspension maintained ata large manufacturing scale. The Sf-9S cell line of the invention iscapable of enhanced expression of recombinant gene products. Thedesignations “Sf-9S” and “ATCC PTA4047” are used herein interchangeably,and refer to the same cell line.

According to one embodiment of the invention, there is provided aprocess for developing cell lines from the parent cell line Sf-9S. Thefirst step of this process involves progressive weaning of cells fromserum-containing media to serum-free media. Master and working cellbanks of the cell line are constructed and qualified according tosafety, identity, and biological criteria or specifications. Prior tocommencement of the clonal selection process, master and working cellbanks of the parental cell line Sf-9 cells are cultivated as monolayercultures for at least 10, preferably at least 20, and more preferably atleast 30 passages in Grace's insect media (Life Technologies, GrandIsland, N.Y. 14072) supplemented with 10% fetal bovine serum (LifeTechnologies, Grand Island, N.Y. 14072). A master cell bank of Sf-9cells is stored in conditioned serum-containing media at −70° C. and inliquid nitrogen. A working cell bank is established from a singlecryovial of the Sf-9 master cell bank and cultivated in serum-containinginsect media for multiple cell passages. The clonal selection process,according to the invention, includes several rounds as demonstrated inFIG. 2.

The method of clonal selection according to the invention describedherein includes generally weaning a plurality of cells fromserum-dependence to obtain at least one cell that can grow in serum-freemedium.

According to another aspect, the invention provides a process forproducing a cell line comprising one or more of the following steps: (a)plating a plurality of cells in wells containing serum-containingmedium, one cell per well; (b) culturing the cell in each separate well;(c) identifying each well with replicating cells; (d) culturing thereplicating cells into replica-plating wells; (e) changing the medium ineach identified well with replicating cells by increasing the proportionof serum-free medium to selum-containing medium; (f) repeatingidentifying, culturing, and medium-changing of steps (c)-(e) until themedium for each well is approximately 100% serum-free; (g) harvestingthe cells from each serum-free well; and (h) culturing the harvestedcells in suspension. Suspension cultures of harvested cells that grow toa predetermined cell density for multiple passages are designatedserum-free cell clones.

According to another embodiment, the method of clonal selection of theinvention includes at least one of the following steps. First, cellclones capable of growing in commercial serum-free media as suspensioncultures are isolated from monolayer cultures of parent Sf-9 cellsdependent on serum-containing media by sequential weaning of parentcells from serum-containing media. According to one embodiment depictedin FIG. 2, Sf-9 parent cells are prepared at step 201 for sequentialserum-weaning. In this embodiment, monolayer and suspension cultures ofSf-9 cells are grown at about 26-28° C. in a dry environment. Shakersuspension cultures are agitated at about 100-150 rpm in a standardorbital or platform shaker incubator and stir flask suspension culturesare stirred at about 25-75 rpm on a standard laboratory magneticstirrer.

In step 202, cell aliquots are dispensed from a cell suspension (onecell per aliquot) of the parental cell line in serum-containing mediainto wells of 96-well dishes at a ratio of one aliquot per well.Following cell attachment, cell acclimation to wells, and exclusion ofwells with no cells and wells with more than one cell, in step 202 themedia is changed from serum-containing media (100%) to a media mixturecomprised of 75% serum-containing media and 25% serum-free media. Instep 203, cells are cultured in “75/25” media mixture for approximatelyone to two weeks. Wells that initially contain only one cell per welland demonstrate cell growth and replication (i.e., four to five cells)after step 203 are subjected in step 204 to another media change to amixture comprising 50% serum-containing media and 50% serum-free media.In step 205, cells in the “50/50” media mixture of step 204 are allowedto grow for approximately another one to two weeks. The media mixture ischanged again in step 206, to a mixture comprising 25% serum-containingmedia and 75% serum-free media. In step 207, cells are allowed to growand replicate in the new “25/75” media mixture of step 206. Afteranother two to four weeks, in step 208 the media is changed in wellscontaining growing cells to a final media comprising serum-free media(100%).

During each step of the weaning process depicted in FIG. 2, a majorityof the cells, for example about 95% of the cells or more, do not survivethe reduction in serum. While not wanting to be bound by this theory, itis believed that this high level of cell death creates a selectivepressure to permit development of a new cell phenotype. In step 209,cells from wells that demonstrate continuous cell growth and replicationare harvested by vigorous aspiration with serum-free media. In step 210,the harvested cells are seeded into larger culture flasks (i.e., 75 or150 cm² T-flasks), and, in step 211, the suspension cultures are grown.When greater than 4×10⁶ cells with a viability >95% is obtained, cellsare harvested in step 211 and seeded into shaker or stir flasks assuspension cultures with a starting cell density, for example, about0.2-0.5×10⁶ cells/ml and a minimal ratio, for example, about 2.5 fortotal vessel capacity to total volume of culture media. In step 212,cell clones that grow exponentially to a saturation cell density ofgreater than 6×10⁶ cells/ml in serum-free media are selected, expanded,and frozen.

Finally, one cell clone is selected, passaged for at least 10,preferably at least 20, and more preferably more than 30 times as asuspension culture in serun-free media at a split ratio of at least1:10, and established as a cell line. This serum-independent cell lineis used to establish a master cell bank and subsequent working cellbanks.

1.1 Sf-9S Cells

According to another aspect of the invention described herein, there areprovided host cells that express one or more recombinant gene productswith an enhanced yield. Insect host cells include, for example,Lepidopteran insect cells, and particularly preferred are Spodopterafrugiperda, Bombyx mori, Heliothis virescens, Heliothis zea, Mamestrabrassicas, Estigmene acrea or Trichoplusia insect cells. Non-limitingexamples of insect cell lines include, for example, Sf21, Sf9, High Five(BT1-TN-5B1-4), BT1-Ea88, Tn-368, mb0507, Tn mg-1, and Tn Ap2, amongothers.

In addition to the serum-weaning process described above, the Sf-9Scells of the present invention have undergone a recombinant peptidesecretion selection process. An example of the process of therecombinant peptide secretion selection, according to the invention, isdemonstrated in FIG. 3. The Sf-9S cells express extracellularly aforeign recombinant protein with an enhanced yield.

According to one embodiment of the invention, the cells are infectedwith a recombinant Baculovirus vector to express recombinant proteins orpolypeptides of medical, pharmaceutical, or veterinary importance.Baculoviruses including Autographa californica multinucleocapsid nuclearpolyhedrovirus (AcMNPV) are propagated in cell lines derived from larvaltissues of insects of the Lepidopteran insect family. General methodsfor handling and preparing baculovirus vectors and baculovirus DNA, aswell as insect cell culture procedures, are outlined for example inO'Reilly et al., 1994; Vaughn, J. 1999, supra; Frieson et a l 986. In:The Molecular Biology of Baculoviruses, Doerffer et al., Eds.Springer-Verlag, Berlin, pages 31-49; Kool et al, 1993. Arch. Virol.130: 1-16, incorporated herein by reference in their entirety.

In one embodiment, polynucleotide molecules, including chimeric andheterologous polynucleotides, which encode a foreign peptide ofinterest, are inserted into the baculovirus genome operably coupled toor under the control of the polyhedrin or other Baculovirus promoters.The recombinant baculovirus vector is then used to infect a host cell.The foreign peptide or protein is expressed upon culture of the cellsinfected with the recombinant virus.

In another embodiment, the invention provides a method for producing aselected foreign protein in an insect cell. The method comprisespreparing infected insect cells that express at least a firstrecombinant viral protein, and infecting the cell with a baculoviruscomprising an expression vector that encodes a second recombinant viralprotein. The first, or the second viral proteins, or both are, forexample, viral capsid proteins including heterologous peptides andchimeric peptides. The cells produced according to the method disclosedherein produce substantially high yields of recombinant baculovirusesexpressing the desired recombinant peptides.

The insect cells of the invention have passed through a recombinantpeptide secretion selection. As described herein, the process ofrecombinant peptide secretion selection includes one or more of thefollowing steps. Cells from a serum-weaned clone are infected with afirst baculovirus expressing a first recombinant protein. Cells capableof secreting high levels of the first recombinant protein are selectedfurther for infection with a second baculovirus expressing a secondrecombinant protein. Cells from a clone that secretes high levels ofboth recombinant proteins independently are passaged further toestablish the Sf-9S cell line of the present invention.

According to a preferred embodiment, the first recombinant protein orthe second recombinant protein, or both, is a viral capsid protein thatself-assembles into virus-like particles. In a more preferredembodiment, the virus-like particles are derived from viral capsidproteins of an enveloped virus, or a non-enveloped virus, including, butnot limited to, an influenza virus, a hepatitis C virus, a retrovirussuch as a human immunodeficiency virus, a calicivirus, a hepatitis Evirus, a papillomavirus, or a combination thereof. In a most preferredembodiment of the invention, the virus-like particles are derived fromhuman papillomavirus.

According to a preferred embodiment of the invention, the Sf-9S cellssupport intracellular, and preferably extracellular, expression ofrecombinant proteins and macromolecules. More preferably, infected Sf-9Scells extracellularly express viral capsid proteins that self assembleinto VLPs. Virus-like particles typically self assemble in the cell andremain intracellular; therefore isolation of these particles requiresprocesses of cell disruption and protein solubilization with theaccompanying risks of VLP disruption, proteolysis and contamination ofthe end product. Accordingly, the infected cells of the invention thatafford self-assembly of viral capsid antigens into VLPs and facilitatesecretion of VLPs extracellularly are highly desirable.

An example of a process for recombinant peptide secretion selection asdepicted in FIG. 3 is described below. FIG. 3 demonstrates thatadditional rounds of clonal selection are used to obtain cells capableof enhanced secretion of recombinant proteins. In step 301, cellaliquots from a cell suspension (one cell per aliquot) of the parentserum-free cell clone (ie., a cell line from one of the serum-free cellclones selected in step 213 of FIG. 2) are replica-plated into each wellof 96-well plates at a ratio of one cell per well. In step 302, wellscontaining a single cell from the original seeding are identified andgrown to confluency. Upon confluency, cells from wells identified assingle cell wells are subcultured into replica plates in step 303. Cellsin replica plates are grown at step 304 to confluency and infected witha first recombinant baculovirus expressing first virus capsid proteinsthat are capable of self-assembly into virus-like particles.

During baculovirus infection, in step 305 the infected cells andextracellular media are harvested by centrifugation to isolate infectedcells and extracellular media, heat-denatured under reduced conditions(>75° C. for 5 minutes in 1% sodium dodecyl sulfate (SDS) and 10 mMβ-mercaptoethanol), and analyzed by SDS-PAGE and Western blot analyseswith antisera to viral capsid proteins. In step 306, cells in replicaplates that contain cell clones exhibiting extracellular VLPs at levelshigher than control Sf-9 cells are infected with a second baculovirusexpressing the second viral capsid proteins that self-assemble intovirus-like particles. The infected cells and extracellular media fromthe second selection round are isolated in step 307 by centrifugationand analyzed by SDS-PAGE and Western blot analyses. The first and secondviral capsid proteins are the same or different proteins and include,for example, rotavirus VP2, VP6, and HPV-16 L1, HPV-L2 proteins, amongothers.

The test results from the first and second rounds of selection (i.e.virus infections producing VLPs) are examined in step 308. The cellclone exhibiting the highest levels of extracellular VLPs from bothvirus infections is chosen in step 310. From the replica plate, cells ofthe selected cell clone exhibiting highest extracellular VLP levels arepassaged repeatedly in suspension culture with serum-free insect cellmedia to establish a cell line. The cell line supports high levels ofextracellular VLP production upon infection with recombinantbaculoviruses expressing viral capsid proteins that self-assemble intovirus-like particles. Thus, in one embodiment, the clone selected instep 310 is processed again according to steps 304-309 with recombinantbaculovirus expressing HPV-16 L1 capsid proteins. The cell clone thatproduces the highest levels of extracellular VLPs for both sets of viralcapsid proteins is chosen in step 311 to establish a cell line capableof producing extracellular VLPs.

Master cell banks of Sf-9S cells are established, for example, from asingle cell passage of the new cell line grown in suspension culture ofserum-free medium and stored at −70° C. in liquid nitrogen in acryopreservation freezing media containing fresh serum-free media,conditioned serum-free media, and dimethyl sulfoxide. Working cell banksare developed, for example, from single cryovials of the master cellbank, subjected to safety and biological testing for qualification as ahost cell substrate for manufacturing of recombinant protein products,and stored at −70° C. in liquid nitrogen in cryovials incryopreservation freezing media as described above.

The Sf-9S cell line of the present invention demonstrate one or more ofthe following properties: (1) they replicate in serum-free media; (2)they are genetically distinct from parent Sf-9 parent cell line; (3)they grow as single cells in suspension cultures; (4) they demonstratecell division rate of approximately 18-24 hours; (5) they demonstratehigh cell viability (more than 95%) upon continuous cell culture formore than one year; (6) they constitute a cell substrate for Autographacalifornica baculoviruses to produce high-titered virus stocks (morethan 10⁷ plaque forming units (pfu)/ml); (7) they are suitable forrecombinant protein expression and production from baculovirus vectors;(8) they are suitable host cell substrates for agarose plaque assays totiter baculovirus stocks; (9) they are compliant with recognizedidentity and safety guidelines; (10) they are suitable cell substratesfor large-scale manufacturing of human and animal biological productsincluding vaccines, therapeutics, and diagnostic reagents; (11) they aresuitable cell substrates for transfection of genes in recombinantbaculovirus transfer vectors and/or bacmids to produce recombinantbaculoviruses, and (12) they produce high levels of extracellular VLPsfrom baculoviruses expressing viral capsid proteins that self-assembleinto VLPs of non-enveloped viruses such as rotaviruses, caliciviruses,hepatitis E virus, and human papillomaviruses and of enveloped virusessuch as influenza virus, hepatitis C virus, and human immunodeficiencyvirus.

In FIG. 4, a confluent monolayer of Sf-9S cells grown in serum-freeinsect cell media is shown at 400× magnification using a phase-contrastmicroscope. The cuboidal and fibroblastic cell morphologies of the cellline are displayed. The cell morphology of Sf-9S cells changes fromfibroblastic to cuboidal, as the monolayer becomes confluent.

Safety testing of the Sf-9S cell line produced according to the presentinvention and deposited at the ATCC may be performed in accordance withUnited States federal regulatory guidelines and include microbialsterility, mycoplasma and spiroplasma growth, endotoxins, adventitiousagents (in vitro and in vivo assays), and electron microscopicexamination for type C endogenous retrovirus particles. The cellidentity of the Sf-9S cell line was shown by karyology and isotypeenzyme analyses, to be S. frugiperda insect species with the typicalpolyploid chromosomal pattern distinct from mammalian cells.

Expression Systems

The expression vector of the invention is a baculovirus vector. Forbaculovirus vectors and baculovirus DNA, as well as insect cell cultureprocedures, see, for example in O'Reilly et al. 1994, incorporatedherein by reference in its entirety. The baculovirus vector construct ofthe invention preferably contains additional elements, such as an originof replication, one or more selectable markers allowing amplification inthe alternative hosts, such as yeast cells and insect cells.

Host cells are infected, transfected, or genetically transformed toincorporate codon-optimized polynucleotides and express polypeptides ofthe present invention. The recombinant vectors containing apolynucleotide of interest are introduced into the host cell by any of anumber of appropriate means, including infection (where the vector is aninfectious agent, such as a viral or baculovirus genome), transduction,transfection, transformation, electroporation, microprojectilebombardment, lipofection; or a combination thereof. A preferred methodof genetic transformation of the host cells, according to the inventiondescribed herein, is infection.

In certain embodiments, there are provided baculovirus vectors thatcontain cis-acting control regions effective for expression in a hostoperatively linked to the polynucleotide to be expressed. Appropriatetrans-acting factors are either supplied by the host, supplied by acomplementing vector or supplied by the vector itself upon introductioninto the host. Host cells are infected with baculovirus vectorscomprising codon optimized polynucleotides to express polypeptides.

The polynucleotides are introduced alone or with other polynucleotides.Such other polynucleotides are introduced independently, co-introducedor introduced joined to the polynucleotides of the invention Thus, forinstance, a polynucleotides (i.e., L1 gene) is tansfected into hostcells with another, separate polynucleotide (i.e., L2 or fusion L2genes) using standard techniques for co-transfection and selection. Inanother embodiment, the polynucleotides encoding L1 capsid protein andthe polynucleotides encoding L2 protein or an L2 fusion protein arepresent on two mutually compatible baculovirus expression vectors whichare each under the control of their own promoter.

3. Codon-Optimized Polynucleotides Encoding HPV Polypeptides

This invention also encompasses nucleic acid sequences that correspondto, and code for the HPV polypeptides. Nucleic acid sequences aresynthesized using automated systems well known in the art. Either theentire sequence is synthesized or a series of smaller oligonucleotidesare made and subsequently ligated together to yield the full-lengthsequence. Alternatively, the nucleic acid sequence is derived from agene bank using oligonucleotides probes designed based on the N-terminalamino acid sequence and well known techniques for cloning geneticmaterial.

In addition, the codon-optimized polynucleotides comprising unusualbases, such as inosine, or modified bases, such as tritylated bases of8-amino adenine bases, to name just a few are polynucleotides, the termis used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term “codon-optimizedpolynucleotide”, as it is employed herein, embraces such chemically,enzymatically or metabolically modified forms of polynucleotide.

The codon-optimized polynucleotides of the present invention encode, forexample, the coding sequence for the mature polypeptide, the codingsequence for the mature polypeptide and additional coding sequences, andthe coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences. Examples of additional coding sequences include,but are not limited to, sequences encoding a leader or secretorysequence, such as a pre-, pro-, or prepro-protein sequences. Examples ofadditional non-coding sequences include, but are not limited to, intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription and mRNAprocessing, including splicing and polyadenylation signals, for example,for ribosome binding and stability of mRNA.

The codon-modified polynucleotides also encode a polypeptide which isthe mature protein plus additional amino or carboxyl-terminal aminoacids, or amino acids interior to the mature polypeptide (when themature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may facilitate protein trafficking, may prolong or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. The additional amino acids may beprocessed away from the mature protein by cellular enzymes.

In sum, a codon-optimized polynucleotide of the present inventionencodes, for example, a mature protein, a mature protein plus a leadersequence (which may be referred to as a preprotein), a precursor of amature protein having one or more prosequences which are not the leadersequences of a preprotein, or a preproprotein, which is a precursor to aproprotein, having a leader sequence and one or more prosequences, whichgenerally are removed during processing steps that produce active andmature forms of the polypeptide.

According to one embodiment of the invention, there are providedcodon-optimized polynucleotides that encode one or more foreignproteins. In this embodiment, the codon optimization of the invention isbased on the following criteria: (1) abundance of aminoacyl-tRNAs for aparticular codon in Lepidopteran species of insect cells for a givenamino acid as described by Levin and Whittome (2000), (2) maintenance ofGC-AT ratio in L1 gene sequence is approximately 1:1, (3) minimalintroduction of palindromic or stem-loop DNA structures, and (4) minimalintroduction of transcription and post-transcription repressor elementsequences.

The optimized genes sequence is synthesized in vitro, for example, asoverlapping oligonucleotides, cloned, and expressed in a host cell.Cloning and expression of the codon modified viral genes were achievedfollowing the methods known in the art and exemplified at Examples 3 and4 herein.

In a preferred embodiment of the invention, polynucleotides encoding aviral gene, for example HPV genes, are optimized for expression in abaculovirus-infected insect cell, comprising one or more of thefollowing steps (a) replacing nucleotide sequences of codons in the genethat are underutilized in insect cells of Lepidopteran species withsequences of preferred codons in insect cells; and (b) for each aminoacid encoded by this modified nucleotide sequence, if a plurality ofcodons for the same amino acid is preferred in insect cells, then thenucleotide sequence of the modified gene is changed farther by selectinga codon from preferred codons for a amino acid so that (i) the ratio ofGC nucleotides to AT nucleotides in the sequence trends toward 1:1; (ii)the number of palindromic and stem-loop structures is minimized unlessindicated otherwise for functional activity; and (iii) the number oftranscription and/or post-transcription repressor elements in thesequence is minimized.

This method was used to develop the codon-optimized polynucleotidesencoding HPV L1 (used to generate L1 VLPs), and HPV L2 (includingwildtype L2), L2/E7, L2/E7/E2, and L2/E6 (used to generate chimericVLPs). The nucleic acid sequences of the codon optimized HPV L1, HPV L2,and HPV L2 fusion genes are represented herein as HPV L1 (SEQ ID NO. 1),HPV L2 (SEQ ID NO. 2,), HPV L2/E7 (SEQ ID NO. 3), HPV L2/E7/E2 (SEQ IDNO. 4), and BPV L2/E6 (SEQ ID NO. 5), respectively.

The method of codon optimization of the invention, as described herein,is used to, inter alia, optimize the expression of variety of envelopedand non-enveloped viral genes expressed in insect cells.

The codon-optimized polynucleotides of the invention include“variant(s)” of polynucleotides, or polypeptides as the term is usedherein. Variants include polynucleotides that differ in nucleotidesequence from another reference polynucleotide. Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical. Asnoted below, changes in the nucleotide sequence of the variant amy besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype, a variant will encode a polypeptide with the same amino acidsequence as the reference.

Changes in the nucleotide sequence of the variant may alter the aminoacid sequence of a polypeptide encoded by the reference polynucleotide.Such nucleotide changes may result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence. According to a preferred embodiment of theinvention, there are no alterations in the amino acid sequence of thepolypeptide encoded by the codon optimized polyneucleotide of theinvention, as compared with the amino acid sequence of the wild typepeptide.

The present invention further relates to polynucleotides that hybridizeto the herein-described sequences. The term “hybridization understringent conditions” according to the present invention is used asdescribed by Sambrook et al., Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory Press 1.101-1.104, 1989. Preferably, astringent hybridization according to the present invention is given whenafter washing for an hour with 1% SSC and 0.1% SDC at 50° C., preferablyat 55° C., more preferably at 62° C., most preferably at 68° C. apositive hybridization signal is still observed. A polynucleotidesequence which hybridizes under such washing conditions with thenucleotide sequence shown in any sequence disclosed herein or with anucleotide sequence corresponding thereto within the degeneration of thegenetic code is a nucleotide sequence according to the invention.

The codon-optimized polynucleotides of the invention includepolynucleotide sequences that have at least about 50%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, 99% or more nucleotide sequence identity to thecodon optimized polynucleotides or a transcriptionally active fragmentthereof. To determine the percent identity of two amino acid sequencesor two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (i.e., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond nucleic acid sequence). The amino acid residue or nucleotides atcorresponding amino acid or nucleotide positions are then compared. Whena position in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % identity=# ofidentical overlapping positions/total # of positions×100). In oneembodiment, the two sequences are the same length.

The determination of percent identity between two sequences also can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST program of Altschul, et al.,1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program program, score 100, wordlength=12 toobtain nucleotide sequences homologous to a nucleic acid molecules ofthe invention. The BLAST protein searches can be performed with theXBLAST program, score 50, wordlength=3to obtain amino acid sequenceshomologous to a protein molecule of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402.

Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST and PSI-Blast programs, the default parameters ofthe respective programs (i.e., XBLAST and NBLAST program can be used(see, HTTP://WWW.NCBI.NLM.NIH.GOV). Another preferred, non-limitingexample of a mathematical algorithm utilized for the comparison ofsequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences of a PAM120 weight residue table, a gap length penalty of 12 and a gap penaltyof 4 can be used. In an alternate embodiment, alignments can be obtainedusing the NA_MULTIPLE_ALIGNMENT 1.0 program, using a GapWeight of 5 anda GapLengthWeight of 1.

4. Recombinant HPV Polypeptides

In general, as used herein, the term polypeptide encompasses variety ofmodifications, particularly those that are present in polypeptidesexpressed by polynucleotides in a host cell. It will be appreciated thatpolypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids, and thatmany amino acids, including the terminal amino acids, may be modified ina given polypeptide, either by natural processes, such as processing andother post-translational modifications, or by chemical modificationtechniques.

It will be appreciated, that polypeptides are not always entirelylinear. For instance, polypeptides may be branched as a result ofubiquitination, and they may be circular, with or without branching,generally as a result of posttranslational events, including naturalprocessing event and events brought about by human manipulation which donot occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translational natural processesand by entirely synthetic methods, as well.

Modifications occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side chains and the amino or carboxyl termini.Blockage of the amino or carboxyl group in a polypeptide, or both, by acovalent modification, occur in a natural or synthetic polypeptides andsuch modifications may be present in polypeptides of the presentinvention, as well. In general, the nature and extent of themodifications are determined by the host cell's post-translationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. It will be appreciated that the sametype of modification may be present in the same or varying degrees atseveral sites in a polypeptide.

The recombinant foreign polypeptide according to the invention includestruncated and/or N-terminally or C-terminally extended forms of thepolypeptide, analogs having amino acid substitutions, additions and/ordeletions, allelic variants and derivatives of the polypeptide, so longas their sequences are substantially homologous to the native antigenicviral polypeptide.

Specifically, as will be appreciated by those skilled in the art, therecombinant viral polypeptides of the invention include thosepolypeptides having slight variations in amino acid sequences or otherproperties. Such variations may arise naturally as allelic variations,as disclosed above, due to genetic polymorphism, for example, or may beproduced by human intervention (i.e., by mutagenesis of cloned DNAsequences), such as induced point, deletion, insertion and substitutionmutants. Minor changes in amino acid sequence are generally preferred,such as conservative amino acid replacements, small internal deletionsor insertions, and additions or deletions at the ends of the molecules.

Substitutions may be designed based on, for example, the model ofDayhoff, et al., Atlas of protein Sequence and Structure, Nat'l Biomed.Res. Found. Washington, D.C., 1978. These modifications can result inchanges in the amino acid sequence, provide silent mutations, modify arestriction site, or provide other specific mutations. The recombinantviral polypeptides may comprise one or more selected antigenicdeterminants of the viral polypeptide peptides, possess catalyticactivity exhibited by their native protein or alternatively lack suchactivity.

The conserved and variable sequence regions of a viral polypeptide andthe homology thereof can be determined by techniques known to theskilled artisan, such as sequence alignment techniques. For example, thedetermination of percent identity between two sequences can also beaccomplished using a mathematical algorithm, as described above.

4.1 Virus-Like Particles (VLPs)

Virus-like particles (VLPs) are the expressed product of thecodon-optimized polynucleotides of the invention. The capsid proteinencoded by the codon-optimized polynucleotide of the invention iscapable of self assembly into virus-like particles that exhibitconformational antigenic epitopes capable of eliciting neutralizingantibodies in a subject.

Encompassed within the scope of the invention are VLPs comprising capsidprotein of non-enveloped and enveloped viruses, including rotaviruses,caliciviruses, hepatitis E virus, and human papillomaviruses, influenzavirus, hepatitis C virus, and retrovirus, including humanimmunodeficiency virus. Preferably, the VLPs comprise Papillomavirus L1capsid protein.

Also encompassed within the scope of the invention are VLPs derived fromdifferent species and genotypes of papillomaviruses. Papillomaviruses ofthe invention are, for example, from human, simian, bovine, or otherorigins. Preferably, the papillomavirus of the invention is a humanpapillomavirus (HPV). More than 100 different human papillomavirus (HPV)genotypes have been isolated. Human papillomavirus genotypes include,but are not limited to, HPV-16, HPV-18, and HPV45 for high-risk cervicalcancers, HPV-31, HPV-33, HPV-35, HPV- 51, and HPV-52 forintermediate-risk cervical cancers, and HPV-6, HPV-11, HPV42, HPV43, andHPV44 for low-risk cervical cancer and anogenital lesions (Bosch et al.,1995; Walboomers et al., 1999). HPV genotypes are also disclosed in PCTpublication No. WO 92/16636 (Boursnell et al., 1992), incorporatedherein by reference in its entirety. HPV-16 is a preferred genotype ofthe invention.

4.2. Chimeric VLPs

Chimeric VLPs refer to viral capsid proteins that encapsulate otherviral proteins or heterologous gene products. A preferred chimeric VLPaccording to the invention is a papillomavirus L1 capsid protein, orpeptide fragment thereof, which encapsulate other papillomavirus geneproducts or heterologous gene products during self-assembly intovirus-like particles. For example, gene products containing the HPV L2,E2, E6, and/or E7 gene products become encapsulated into the HPV L1 VLPsand are considered herein as chimeric VLPs.

4.2.1. Fusion Proteins

As one of skill in the art will appreciate, and as discussed above, theHPV polypeptide of the invention can be fused to heterologouspolypeptide sequences. For example, the HPV L2 polypeptide of thepresent invention (including fragments or variants thereof) may be fusedto one or more additional HPV polypeptide or other non-enveloped virusor enveloped virus polypeptides.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepotypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33, 1997; Harayama, Trends Biotechnol. 16(2):76-82,1998; Hansson, et al., J. Mol. Biol. 287:265-76, 1999; and Lorenzo andBlasco, Biotechniques 24(2):308-13, 1998 (each of these patents andpublications are hereby incorporated by reference in its entirety). DNAshuffling involves the assembly of two or more DNA segments byhomologous or site-specific recombination to generate variation in thepolynucleotide sequence. In another embodiment, polynucleotides of theinvention, or the encoded polypeptides, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of a polynucleotide encoding a polypeptide of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

Nucleic acids encoding the above fusion polypeptides can be recombinedwith a gene of interest as an epitope tag (i.e., the hemagglutinin(“HA”) tag or flag tag) to aid in detection and purification of theexpressed polypeptide. For example, a system described by fanknecht etal. allows for the ready purification of non-denatured fusion proteinsexpressed in human cell lines. (See, for example, Janknecht et al.,Proc. Natl. Acad. Sci. USA 88:8972-897, 1991). In this system, the geneof interest is subcloned into a vaccinia recombination plasmid such thatthe open reading frame of the gene is translationally fused to anamino-terminal tag consisting of six histidine residues. The tag servesas a matrix-binding domain for the fusion protein. Extracts from cellsinfected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

The cloning and expression of the L2 and L2 fusion genes can be achievedfollowing the methods known in the art. One example of such methods isexemplified at Example 4 herein.

5. Production, Isolation and Purification of Recombinant VLPs

The present invention, as disclosed and described herein, providesmethods for production, isolation and purification of recombinant viralgene products that are capable of self-assembly into intact virus-likeparticles exhibiting conformational antigenic epitopes. The virus-likeparticles of the invention can be prepared as pharmaceuticalcompositions or vaccines to induce a high-titer neutralizing antibodyresponse in vertebrate animals. The self-assembling capsid proteins canalso be used as elements of diagnostic immunoassay procedures forpapillomavirus infection.

5.1. Production of YLPs

The present invention encompasses process for producing a recombinantvirus-like particle including, for example, a virus-like particle of anenveloped virus or a non-enveloped virus, by infecting a permissiveinsect cell with a recombinant baculovirus that encodes viral capsidand/or envelope genes of one or more viruses. In one embodiment, theinvention provides methods for harvesting and purifying HPV VLPs,including HPV chimeric VLPs from infected insect cells or other hostcells. The baculovirus-infected cell expresses viral capsid and/orenvelope proteins that self-assemble into virus-like particles. The VLPsare expressed intracellularly, extracellularly, or both.

In a preferred embodiment of the invention, the VLPs are producedextracellularly.

According to another embodiment of the invention, there is provided amethod for the production of intracellular and extracellular HPV.VLPs.In this embodiment as depicted step 505 of FIG. 5, production ofintracellular and extracellular HPV-16 L1 VLPs begins with highmultiplicity infection of log phase Sf-9S cells with an aliquot of aworking virus stock, as depicted in step 504 of FIG. 5, of baculovirusesexpressing HPV L1 capsid proteins. The virus infection can be monitoreddaily by the trypan blue exclusion method for cytopathic effects, cellviability, and cell density and by SDS-PAGE and Western blot analyses ofrecombinant HPV L1 capsid proteins in infected cells and extracellularmedia. At peak recombinant HPV L1 gene expression, infected cells andextracellular media containing intracellular and extracellular HPV L1VLPs, respectively, are harvested and processed to obtain purified HPVL1 VLP products as outlined in step 506 of FIG. 5 and in depicted inmore detail in FIG. 6 described below.

5.2. Production of Chimeric VLPs

According to yet another embodiment of the invention, there is provideda method for production of chimeric VLPs. In a preferred embodiment, thechimeric VLPs are HPV chimeric VLPs. In one embodiment, as depicted inFIG. 5, production of intracellular HPV chimeric VLPs begins with highmultiplicity infection of log phase Sf-9S insect cells in suspensioncultures containing serum-free insect cell medium with aliquots of HPVL1 and L2 fusion working virus stocks (i.e., recombinant baculovirusesexpressing L1 capsid protein and L2 fusion protein, respectively).

The ratio of co-infecting viruses is approximately at least about 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, or more. In a preferred embodiment the ratio of co-infectingviruses is approximately at least about 1:3, 1:5 or 1:10 (L1 to L2virus). The virus infection is monitored daily by the trypan blueexclusion method for cytopathic effects, cell. viability, and celldensity and by SDS-PAGE and Western blot analyses of recombinant HPV L1and L2 fusion proteins in infected cells. At peak recombinant HPV L1 andL2 fusion gene expression, infected cells containing intracellular HPVchimeric VLPs are harvested and processed to obtain purified HPVchimeric VLPs products as outlined in FIG. 5 and described in moredetail below with reference to FIG. 7. This co-infection process may beused to manufacture recombinant papillomavirus chimeric VLPs of variousviral genotypes including, but not limited to, those identified aboveassociated with HPV infections and cancers.

5.3 Upstream Processing of L1 VLPs

The present invention also includes methods for upstream processing oftransformed host cells including yeast, insect, and mammalian cellsexpressing recombinant VLPs. The recombinant VLPs are preferably L1VLPs. More preferably the recombinant VLPs are HPV L1 VLPs.

In one embodiment, the process for purifying recombinant extracellularHPV L1 VLPs includes at least one of the steps of harvesting a cellsuspension containing recombinant extracellular HPV L1 VLPs to produce aharvested supernatant, clarifying the harvested supernatant,concentrating the clarified supernatant, and dialyzing the concentratedsupernatant.

In another embodiment, the process for purifying intracellularrecombinant HPV L1 VLPs includes at least one of the steps of harvestinga cell suspension comprising infected cells containing intracellularrecombinant papillomavirus L1 VLPs, and disrupting the harvested cells(which may have been resuspended in a buffer containing proteaseinhibitors) by, for example, sonication to produce crude infected celllysates containing recombinant HPV L1 VLPs. The crude infected celllysates is then clarified by, for example, centrifugation to produce aclarified supernatant containing recombinant HPV L1 VLPs. The clarifiedsupernatant is then concentrated by, for example, ultrafiltration toproduce a concentrate containing recombinant HPV L1 VLPs, and dialyzingconcentrates, for example, against high salt buffers by ultrafiltrationto produce a diafiltered crude product containing recombinant HPV L1VLPs. The crude product is further processed by downstream processing asdescribed below.

One example of the upstream processing of L1 VLPs is shown by FIG. 6.Recombinant HPV L1 VLPs from baculovirus-infected insect cells (step601) or from other cells expressing recombinant HPV VLPs are harvestedby, for example, low-speed centrifugation (step 602). In step 603 of theembodiment as depicted in FIG. 6, extracellular supernatants areseparated from cell pellets by, for example, aspiration followingcentrifugation and, at step 604, are clarified by, for example,centrifugation to remove large cell debris from extracellularrecombinant HPV L1 VLPs. At step 605 of the embodiment depicted in FIG.6, cell pellets containing intracellular recombinant HPV VLPs arediscarded or resuspended in multifold cell volumes of buffer solutionsuch as phosphate-buffered saline solution to produce a cell suspension.In step 606 of the embodiment depicted in FIG. 6, resuspended cellscontaining intracellular recombinant HPV L1 VLPs are disrupted with, forexample, several pulses of sonication to produce crude cell lysateswithout proteosome disruption. The cell sonicates containing HPV L1 VLPsare monitored for cell disruption by, for example, the trypan blueexclusion method. In step 607 of the embodiment depicted in FIG. 6,crude cell lysates containing intracellular BPV L1 VLPs are clarifiedby, for example, centrifugation to remove large cellular debris.

In another embodiment, clarified supernatants from supernatant media andcell lysates are combined as depicted in step 608 and concentratedmultifold by, for example, ultrafiltration using hollow fiber filters(step 609). The concentrates are dialyzed against multiple volumes ofbuffer by, for example, ultrafiltration using hollow fiber filters (step610).

Biological products including mock and/or wild type baculovirus-infectedinsect cells as described above may be used to prepare cell lysatescontaining host cells and/or baculovirus proteins. The proteins in thecell lysates are solubilized by, for example, sonication to disruptcells and clarified by centrifugation as described above and mixed withFreund's or other adjuvant to produce immunogens.

Immunogenicity of the immunogens obtained above can be determinedaccording to methods known in the art. For example, the immunogens canbe administered at least once by intramuscular, subcutaneous, orintranasal routes into animals. After testing sera or immune cells fromimmunized animals for antigen specificity, the sera or immune cells fromimmunized animals were isolated following vaccination. The antibodytiter specific for host cell and/or baculovirus proteins were determinedby immunodetection methods such as ELISA or Western blot assays. Thetitered antisera can were used in immunodetection assays such as Westernblot analysis to determine the level of host cell and/or baculoviruspresent in recombinant protein products derived from baculovirus-derivedsources. T cell assays such as lumphocute proliferation assays andELISPOT assays, which are used to determine the abundance of CD8+cytotoxic T cells and the level of accompanying lymphokines andcytokines produced as a result of sensitization with the VLPs in thisinvention following immunization.

5.4 Upstream Processing of Chimeric VLPs

In another embodiment of the invention, methods for upstream processingof expressed VLPs are provided. In a preferred embodiment, the inventiondiscloses the upstream processing of HPV VLPs and chimeric VLPs derivedfrom insect cells co-infected with recombinant baculoviruses expressingL1 and L2 fusion genes.

In a specific embodiment, the upstream processing of chimeric VLPsgenerally includes one or more of the steps of harvesting a cellsuspension comprising co-infected cells containing intracellularrecombinant HPV chimeric VLPs, resuspending the harvested infected cellsin a buffer containing a protease inhibitor, disrupting the resuspendedinfected cells by, for example, sonication, to produce crude infectedcell lysates containing recombinant HPV chimeric VLPs, clarifying thecrude infected cell lysates by, for example, centrifugation, to producea clarified supernatant containing recombinant HPV chimeric VLPs,concentrating the clarified supernatants by, for example,ultrafiltration, to produce a concentrate containing recombinant HPVchimeric VLPs, and dialyzing concentrates against high salt buffers by,for example, ultrafiltration, to produce a diafiltered crude productcontaining recombinant HPV chimeric VLPs. The crude product thusobtained goes through downstream processing.

In a preferred embodiment, upstream processing of the invention isperformed following steps of the method depicted in FIG. 7. RecombinantHPV chimeric VLPs are harvested from transformed cells, for example,baculovirus-infected insect cells, by, for example, low-speedcentrifugation (step 702). At step 703, supernatant media are separatedfrom cell pellets by, for example, aspiration following centrifugation.At step 704 supernatants media are discarded and cell pellets containingintracellular recombinant papillomavirus chimeric VLPs were resuspendedin multiple cell volumes of a buffer solution containingprotease-inhibitors that block the activity of at least one of thefollowing protease classes: serine, aspartate, cysteine, and metallo. Instep 705, resuspended cells containing intracellular recombinant HPVchimeric VLPs are disrupted with, for example, several pulses ofsonication to produce crude cell lysates without proteosome disruptionthat may cause proteolysis of chimeric L2 fusion proteins. The cellsonicates containing HPV chimeric VLPs are monitored for cell disruptionby the trypan blue exclusion method. In step 706, crude cell lysatescontaining intracellular HPV chimeric VLPs were clarified by, forexample, centrifugation to remove large cellular debris. Clarifiedsupernatants from cell lysates are concentrated multifold byultrafiltration using hollow fiber filters (step 707). The concentratesare dialyzed against multiple volumes of buffer containing proteaseinhibitors by ultrafiltration using hollow fiber filters (step 708).

5.5 Dowstream Processing of VLPs

In yet another embodiment of the invention, methods for downstreamprocessing of expressed VLPs are provided. In a preferred embodiment,the invention discloses the downstream processing of HPV VLPs harvestedfrom cells.

Downstream processing as depicted in FIGS. 5 and 8A-8C includes one ormore of the following steps: a linear sucrose gradient scheme (FIG. 8A),a chromatographic scheme (FIG. 8B), or a sucrose step gradient scheme(FIG. 8C). The product of these purification schemes yields recombinantHPV VLPs that are formulated to inactivate residual baculoviruscontaminants by one or more of the following treatments: detergenttreatment to remove process excipients; by ultrafiltration, to provide abuffer solution that promotes VLP stability; by diafiltration, to removeany microbial contaminants by terminal filtration.

5.5.1. Downstream Processing of VLPs: Linear Sucrose Gradient Scheme

The methods of the present invention encompasses downstream processingof crude materials containing recombinant VLPs by one or more of thefollowing steps: sedimenting the recombinant VLPs through a first linearsucrose gradient (for example, by continuous flow ultracentrifugation),collecting fractions from the first gradient, identifying fractionscontaining recombinant VLPs, pooling fractions containing recombinantVLPs, rebanding pooled fractions through a second linear sucrosegradient, collecting fractions from a second gradient, and poolingfractions containing recombinant VLPs from the second gradient. In anembodiment, only one sedimentation step is used.

In one specific embodiment depicted in FIG. 8A, diafiltered concentrates(step 610 of FIG. 6 and step 708 of FIG. 7) or other materialscontaining VLPs are purified by continuous flow rate-zonalultracentrifugation on linear sucrose gradients, based primarily on themass and density of recombinant VLPs in sucrose (FIGS. 5 and 8A). Forexample, diafiltrates containing recombinant HPV VLPs are loaded underpressure onto approximately 0-65% linear sucrose gradients in a verticalrotor accelerating at high speed in a continuous flow ultracentrifuge.The gradient is resolved by ultracentrifugation at high speed untilrecombinant HPV VLPs separate from baculovirus particles (step 802).Gradient materials from the first round of sucrose gradients aremonitored by ultraviolet light during collection in a fractioncollector. Gradient fractions from the first round of linear sucrosegradients are analyzed by SDS-PAGE and Western blot analysis usingantisera against papillomavirus L1 capsid proteins and/or L2 fusionproteins. Peak fractions containing HPV VLPs or their component proteinsare pooled, diluted multifold with buffer solution, and are subjected toa second round of ultracentrifugation on linear sucrose gradients (step803).

In another embodiment, gradient materials from the second round ofsucrose gradients are monitored by ultraviolet light during collectionin a fraction collector. Gradient fractions from the second round ofultracentrifugation may be analyzed also by SDS-PAGE and Western blotanalysis using antisera against papillomavirus L1 capsid proteins and/orL2 fusion proteins. Peak fractions containing HPV VLPs or theircomponent proteins are pooled. The purified recombinant HPV VLPs in thepooled fractions are formulated as recombinant HPV VLP products.

5.5.2. Downstream Processing of VLPs: Chromatographic Scheme

The chromatographic method for downstream processing of VLPs, andpreferably HPV VLPs includes at least one of the following three steps:adsorptive cation exchange chromatography using a pH gradient, affinitychromatography using heparin-like matrices for binding VLPs, anddisplacement anion exchange chromatography.

In the first chromatography step, diafiltered concentrates (step 610 ofFIG. 6 and step 708 of FIG. 7) or other materials containing HPVpapillomavirus VLPs, are loaded in the initial chromatographic step(step 805 of FIG. 8B) onto a chromatography column containing a strongcation exchange chromatography resin with an exposed amino group such asStreamline SP (Amersham Biosciences) that is equilibrated with multiplevolumes of loading buffer at low salt and a pH between 4.5 to 6.0. Thisstep separates recombinant HPV VLPs from the bulk majority of hostcontaminant proteins and other molecules based on the isoelectric chargeof HPV L1 proteins.

Following binding of diafiltrates or other materials containingrecombinant papillomavirus VLPs to the charged resin, the bound columnis washed with multiple volumes of loading buffer. In one embodiment,bound recombinant papillomavirus VLPs are eluted from the column resinusing a low salt pH step gradient from 6.0 to 8.0. Elution fractions areanalyzed by SDS-PAGE and Western blot analysis using antisera againstpapillomavirus L1 capsid proteins. Purification of chimeric VLPs by thischromatographic scheme has not been tried to date. In anotherembodiment, peak fractions containing VLPs or their component proteinsare pooled.

In the second chromatography step (FIG. 8, step 806), the pooled eluatesfrom step 805 resulting from the cation exchange chromatography step orother materials containing recombinant papillomavirus VLPs are dialyzedagainst multiple volumes of affinity loading buffer by diafiltration.The dialysate is loaded onto a column containing heparin agarose orother molecules having an exposed carboxy group such as, for example,heparin sulfate glycans, glycoaminoglycans, α₆β₁ integrin, α₆β₄integrin, syndecan 1, Matrex Cellufine Sulfate (American Biosciences),or other heparin-like resins equilibrated with affinity loading buffer.Affinity chromatography using heparin serves as receptors forpapillomavirus as binding matrices and affords high levels of specificand selective purification of recombinant HPV VLPs.

The bound column is washed with multiple volumes of affinity loadingbuffer. Bound proteins including recombinant papillomavirus VLPs, areeluted from the column resin using a linear salt gradient fromapproximately 300 mM to 2 M. Elution fractions are analyzed by SDS-PAGEand Western blot analysis using antisera against papillomavirus L1capsid proteins and/or L2 fusion proteins. Peak fractions containingVLPs or their component proteins are pooled.

In the third chromatography step, pooled eluates from the affinitychromatography step (step 806), that contain recombinant papillomavirusVLPs are dialyzed against multiple volumes of anion loading buffer bydialfiltration. The removal of small-molecular weight molecules andresidual host contaminant proteins in pooled affinity eluates or othermaterial containing recombinant HPV VLPs is provided by anion exchangechromatography (FIG. 8 step 807) using a displacement polymer as a finalpolishing step based on the isoelectric point of HPV L1 proteins. Thedialysate is loaded onto a column containing a strong anion exchangechromatography resin with an exposed carboxyl group such as, forexample, Q Sepharose Fast Flow (FF) (Amersham Biosciences), ToyopearlSuper Q-650 M (Tosoh Biosep), Q Sepharose FF, or Fractogel TMAE (USB)equilibrated with multiple volumes of anion loading buffer.

Bound proteins including recombinant HPV VLPs are displaced from theanion column resin with a linear gradient from approximately 0 to 5mg/ml dextran sulfate (5000 MW). Elution fractions are analyzed by, forexample, SDS-PAGE and Western blot analysis using antisera againstpapillomavirus L1 capsid proteins. If required, peak fractionscontaining recombinant HPV VLPs or their component proteins are pooledand dialyzed by ultrafiltration against multiple volumes of high saltbuffer to remove dextran sulfate.

5.5.3 Downstream Processing of VLPs: Sucrose Step Gradient Scheme

In yet another alternate embodiment of the present invention,diafiltered concentrates (step 610 of FIG. 6 and step 708 of FIG. 7) orother materials containing VLPs are purified by rate-zonalultracentrifugation on discontinuous sucrose step gradients basedprimarily on mass and density of recombinant VLPs in sucrose.

The methods of the present invention encompasses downstream processingof recombinant VLPs and preferably recombinant HPV VLPs from crudematerials by pelleting crude materials containing recombinant HPV VLPsthrough a sucrose cushion, resuspending the pelleted recombinant HPVVLPs, banding resuspended recombinant HPV VLPs by ultracentrifugation ondiscontinuous linear step gradients, collecting at least one bandscontaining recombinant HPV VLPs, and dialyzing banded material bydiafiltration to remove sucrose.

In one embodiment depicted in FIG. 8C, diafiltrates containingrecombinant HPV VLPs are loaded onto approximately 25% sucrose cushionsin a swinging bucket rotor accelerating at high speed in anultracentrifuge (step 809 of FIG. 8). The pellets at the bottom of thesucrose cushion are collected, while the sucrose cushion and loadmaterial are discarded. The sucrose cushion pellets are solubilized inbuffer and loaded onto sucrose step gradients containing multiple stepscomprising approximately 25 to 65% sucrose. The sucrose step gradientsare resolved by ultracentrifugation in a swinging bucket rotor at highspeed until recombinant HPV VLPs are separated from baculovirusparticles (step 810). Gradient materials from the first round of sucrosestep gradients are monitored by ultraviolet light during collection in afraction collector. Gradient fractions are analyzed by, for example,SDS-PAGE and Western blot analysis using antisera against papillomavirusL1 capsid proteins and/or L2 fusion proteins.

Peak fractions containing HPV VLPs or their component proteins arepooled, diluted multifold with buffer solution, and optionally subjectedto a second round of ultracentrifugation on sucrose step sucrosegradients (step 811). Gradient materials from the second round ofsucrose step gradients are monitored by ultraviolet light duringcollection in a fraction collector. Gradient fractions from the secondround of ultracentrifugation are analyzed by, for example, SDS-PAGE andWestern blot analysis using antisera against papillomavirus L1 capsidproteins and/or L2 fusion proteins. Peak fractions containing HPV VLPsor their component proteins are pooled. In an embodiment, the purifiedrecombinant HPV VLPs in the pooled fractions are formulated asrecombinant HPV VLP products.

5.6. Formulation of Papillomavirus VLP Products

According to the present invention as depicted in FIG. 5, pooledfractions or other material containing recombinant VLPs from thepurification schemes described above may contain recombinant baculovirusparticles, which are inactivated by treatment with, for example, anonionic detergent, surfactants, ultraviolet light, or a combinationthereof. In an embodiment of the present invention, a nonionicdetergent, and a surfactant, such as Triton X-100, is added to. theproduct containing recombinant HPV VLPs at a final concentration ofapproximately >0.1%. The recombinant papillomavirus VLP mixture withdetergent is incubated for at least approximately one hour to inactivateresidual baculoviruses.

In an another embodiment, the recombinant HPV VLPs are irradiated withone or more rounds of ultraviolet (UV) light <300 nm and then incubatedin a nonionic detergent, or both. Multiple log reduction of thebaculovirus is afforded by these treatments which may have additive orsynergistic effect. The process for inactivating residual baculovirusproducts are also used for other recombinant protein products, includingrecombinant protein products comprising VLPs of virus types identifiedabove. In addition, VLP products treated according to this process aredialyzed against a buffer in order to refold the conformational epitopesof the VLPs in the product.

In yet another embodiment, following the baculovirus inactivationtreatment(s), recombinant HPV VLP products are dialyzed byultrafiltration against multiple volumes of high salt buffer containingapproximately >0.5 M sodium chloride at approximately neutral pH toremove process excipients such as sucrose, Triton X-100 detergent, andother molecules. Diafiltrates containing recombinant HPV VLP bulkproducts are filtered aseptically through a 0.2 μm membrane at ambienttemperature to remove microbial contaminants. To maintain high levels ofintact VLPs in the final bulk products, the filtered recombinant IPVVLPs are dispensed directly into sterilized 316 L stainless steel tanks,silanized borosilicate glass bottles, or polyethylene plastic bioprocessbags and stored at 2 -8° C. for <six (6) months, or at <−70° C. for 2years.

Bulk recombinant HPV VLP products made according to the presentinvention are formulated alone or with adjuvants such as, for example,Novasomes™ and micelle nanoparticles, among others. For monovalentproducts, bulk products containing one genotype of recombinant HPV VLPsare diluted with buffer solution to the appropriate antigenconcentration such as 100 μg/ml, mixed with an adjuvant, adjusted forfinal pH and salt concentrations, filtered aseptically through 0.2 μmmembranes, and dispensed into silanized borosilicate vials. Formultivalent products, equal molar antigen concentrations of bulkproducts representing more than one genotype of recombinant HPV VLPs areformulated and processed into final container products as describedabove for monovalent products. Final container products are stored at 2-8° C. for <6 months or <−70° C. for extended time such as two years orless. Following qualification of final container products for purity,strength, identity, potency, and safety, final container products areused as pharmaceutical composition, prophylactic vaccines, or diagnosticreagents.

In one embodiment, prophylactic vaccines for the prevention of analgenital warts are formulated as mixtures of at least HPV-6 and/or HPV-11L1 or chimeric VLPs. Prophylactic vaccines for the prevention ofHPV-induced cervical cancer are formulated as mixtures of at leastHPV-16, HPV-18, HPV-31, and/or HPV-33 L1 or chimeric VLPs. Therapeuticsfor treatment of anal genital warts are formulated as mixtures of atleast HPV-6 and/or HPV-11 chimeric VLPs. Pharmaceutical compositions fortreatment of HPV-induced cervical cancer are formulated as mixtures ofat least HPV-16, HPV-18, HPV-31, and HPV-33 chimeric VLPs.

These and other products comprising recombinant VLPs made according tothe present invention are administered by various parenteral and localroutes including but not limited to intramuscular, intradermal,intranasal, or oral, according to conventional protocols. Reagents usedfor diagnosis of HPV infections and associated neoplasia may beformulated as type-specific products capable of detecting antibodies forone or more genotypes of HPV.

5.7. Characterization of VLP In Final Bulk Products

Immunological identification of recombinant products made according tothe present invention is afforded by, for example, Western blot analysisusing polyclonal sera for HPV L1 capsid antigens (linear epitopes) or byenzyme linked immunoadsorbent assay (ELISA) using monoclonal antiserafor PV L1 conformational epitopes specific for neutralizing antibodies.For Western blot analyses, aliquots (2 μg) of recombinant proteins fromcrude lysates, purified intermediates, or purified VLPs and control L1capsid proteins are heat denatured (5-10 min. at 95-99° C.) underreduced conditions with β-mercaptoethanol (10 mM) and loaded onto 4-12%NuPAGE (Novex) protein gels (FIG. 9) or equivalent polyacrylamide gels.

Proteins are resolved by gel electrophoresis in MES buffer under reducedconditions. Control proteins include recombinant PV L1 capsid proteinsverified for authenticity, host cell proteins, and/or AcMNPV baculovirusproteins. Protein molecular weight markers are, for example, SeeBluepre-stained standards (Novex) including proteins with molecular weightsof 188 kilodaltons (kD), 62 kD, 49 kD, 38 kD, 28 kD, 18 kD, 14 kD, 6 kD,and 3 kD. For protein gels, the electrophoresced proteins are visualizedby staining with Colloidal Coomassie Blue reagent (Novex). The molecularweights of the L1 proteins are 50-65 kD depending on the species andgenotype of papillomavirus capsid gene. The purity of purifiedrecombinant HPV L1 VLPs purified by the invention is expected to be 95%or more as determined by scanning densitometry. No more than 5% of thepurified recombinant HPV L1 VLP product is expected to be proteolyticbreakdown products.

For Western blot analysis (FIGS. 10A -10C, as also described withreference to Example 21, below), proteins are transferred byelectroblotting in methanol from unstained protein gels containing L1capsid proteins and control proteins to nitrocellulose orpolyvinyldifluoride membranes. Bound membranes are reacted with primaryantisera including antisera to PV L1 capsid proteins, polyclonal sera tohost cell proteins, and/or polyclonal sera #3 to AcMNPV wild typebaculovirus proteins. Bound primary antibodies are reacted withsecondary antisera comprised of anti-IgG conjugated to alkalinephosphatase. The bound secondary antibodies are detected by reactingwith the chromogenic substrate such as NBT/BCIP (InVitrogen) or thechemiluminescent substrate Lumi-Phos (In Vitrogen). The anti(α)-papillomavirus L1 sera is expected to detect protein bands withmolecular weights of about 50 to 65 kD depending on the species andgenotype of papillomavirus L1 capsid gene. Less than 5% of therecombinant PV L1 VLP products purified by the invention is expected tobe degradation breakdown products. Less than 5% reactivity is expectedto be seen using antisera to host or vector proteins.

The potency of recombinant HPV VLPs according to the present inventionare ascertained by, for example, ELISA testing using antibodies specificfor conformational epitopes on papillomavirus L1 capsid proteins thatelicit neutralizing antibodies. In one embodiment of the presentinvention, ELISA testing of recombinant HPV-16 L1 VLP bulk products isperformed using murine monoclonal antibody H16.V5 (Christensen et al.,1996). Dilutions of VLPs (antigen) and control proteins such as VLPs,denatured VLPs, and heterologous proteins are bound to wells of ELISAplates, and a constant amount of monoclonal antibody is added to eachwell.

Antigen-antibody binding occurs for at least two time durations, such asone minute and 2.5 hours. Antigen-antibody complexes are washedsuccessively with wash buffer to remove nonspecific antigens. Asecondary antibody comprised of anti-murine immunoglobulins conjugatedto an enzyme such as horseradish peroxidase is added to each well of theELISA plate. Detection of antigen-antibody complexes is afforded by theaddition of a chromogenic substrate such as NBT/BCIP (InVitrogen).

As depicted in FIG. 11, and also described with reference to Example 20,below, the L1 proteins of Triton-treated recombinant HPV VLPs madeaccording to the methods of the present invention are not degraded, asdetermined by SDS-PAGE and Westernblot analyses. Triton-treatedrecombinant HPV VLPs remain as intact VLPs, as determined by analyticalsize exclusion chromatography and/or electron microscopy. Theconformational epitopes of Triton-treated recombinant HPV VLPs madeaccording to the methods of the present invention are restored bydiafiltration against 0.5 M sodium chloride buffers, as determined byELISA analysis using monoclonal antibodies raised against neutralizingepitopes of L1 antigens such as H1I6.V5 monoclonal antibody.

The amount of total protein purified by the methods of the presentinvention are determined by one of several calorimetric methods such asthe bicinchoninic acid (BCA) assay or other protein quantitation assayby one skilled in the art of protein chemistry. The absolute amount ofprotein are determined by acid hydrolysis and amino acid determination.The results are compared with those results from colorimetric assays toadjust the relative amounts.

The amount of intact VLPs present following purification of recombinantHPV VLPs according to the present invention are ascertained byanalytical size exclusion chromatography and/or electron microscopy.Size exclusion chromatography (SEC) are used to assess the relativeamount of VLPs in production lots of HPV VLPs and and the relativeamount of other viral VLPs made according to the present invention. Inone embodiment, the pre-poured column used for SEC HPLC is an analyticalsize-exclusion HPLC column such as a TSK-GEL G6000PWXL column (TosohBiosep) that is used with, for example, a fractionation range of morethan 1,000,000 daltons to approximately 20,000 daltons. The test sampleis applied to the column with a resolution for intact VLPs atapproximately 15-16 minutes and monomeric proteins at approximately24-26 minutes.

Other macromolecules such as capsomeres of pentameric HPV L1 structuresare resolved at 19-20 minutes when present. An analytical HPLC systemsuch as a Waters 6000 HPLC system using Millennium computer softwareprovides the mechanics and programs necessary for sample injection,buffer transfer, column development, UV monitoring, fraction collection,and protein data management. Data is presented in a graphic format withprotein absorbance as a function of column development in minutes, asexemplified by FIG. 12 and also described with reference to Example 20,below. Confirmation of SEC EPLC results on viral VLPs is obtained bynegative-stain electron microscopy EM). Purified recombinant HPV VLPsare adsorbed onto carbon coated transmission electron microscopy (TEM)grids, stained with 1% uranyl acetate, and examined with a Philipselectron microscope at 36,000× magnification. Results are shown in FIG.13, also described with reference to Example 20, below. The size of theHPV-16 L1 VLPs is estimated about 40-55 nm.

6. Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising a therapeutically effective amount of one or more recombinantviral gene products, VLPs, agonists, antagonists, or a biologicallyactive fragment of a viral gene product. The recombinant papillomavirusgene products preferably comprise HPV VLPs. More preferably, VLPs areHPV L1 VLPs, or chimeric VLPs. Administration of the pharmaceuticalcompositions of the invention, including vaccines, results in adetectable change in the physiology of a recipient subject, preferablyby enhancing a humoral or cellular immune response to one or morepapillomavirus antigens.

A multivalent vaccine of the present invention can confer protection toone or more genotypes of papillomavirus. The present invention thusconcerns and provides a means for preventing or attenuating infection byat least one papillomavirus genotype. As used herein, a vaccine is saidto prevent or attenuate a disease if its administration to an individualresults either in the total or partial attenuation (i. e,. suppression)of a symptom or condition of the disease, or in the total or partialimmunity of the individual to the disease.

The “protection” provided need not be absolute, i.e., the papillomavirusinfection need not be totally prevented or eradicated, provided thatthere is a statistically significant improvement relative to a controlpopulation. Protection can be limited to mitigating the severity orrapidity of onset of symptoms of the disease.

The pharmaceutical preparations of the present invention, suitable forinoculation or for parenteral or oral administration, are in the form ofsterile aqueous or non-aqueous solutions, suspensions, or emulsions, andcan also contain auxiliary agents or excipients that are known in theart. The pharmaceutical composition of the invention can furthercomprise immunomodulators such as cytokines which accentuate the immuneresponse. (See, i.e., Berkow et al., eds:. The Merck Manual, FifteenthEdition, Merck and Co., Rahway, N.J., 1987; Goodman et al., eds.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, EighthEdition, Pergamon Press, Inc., Elmsford, N.Y., 1990; Avery's DrugTreatment: Principles and Practice of Clinical Pharmacology andTherapeutics, Third Edition, ADIS Press, LTD., Williams and Wilkins,Baltimore, Md., 1987; and Katuung, ed. Basic and Clinical Pharmacology,Fifth Edition, Appleton and Lange, Norwalk, Conn., 1992, whichreferences and references cited therein, are entirely incorporatedherein by reference as they show the state of the art.

As would be understood by one of ordinary skill in the art, when acomposition of the present invention is provided to an individual, itcan further comprise at least one of salts, buffers, adjuvants, or othersubstances which are desirable for improving the efficacy of thecomposition. Adjuvants are substances that can be used to specificallyaugment at least one immune response. Normally, the adjuvant and thecomposition are mixed prior to presentation to the immune system, orpresented separately.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, mannitol, sorbitol, trehelose, andthe like. The composition, if desired, can also contain minor amounts ofwetting or emulsifing agents, or pH buffering agents. These compositionscan take the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations and the like.

The pharmaceutical composition of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with anions such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with cations suchas those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

Adjuvants can be generally divided into several groups based upon theircomposition. These groups include lipid micelles, oil adjuvants, mineralsalts (for example, AlK(SO₄), AINa(SO₄)₂, AlNH₄(SO₄)), silica, kaolin,polynucleotides (for example, poly IC and poly AU nucleic acids), andcertain natural substances, for example, wax D from Mycobacteriumtuberculosis, substances found in Corynebacterium parvum, or Bordetellapertussis. Preferred adjuvant of the invention includes, for example,Freund's adjuvant (DIFCO), alum adjuvant (Alhydrogel), MF-50 (Chiron)Novasomes™, or micelles, among others.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a “therapeutically or prophylacticallyeffective amount” if the amount administered is physiologicallysignificant.

The pharmaceutical composition of the invention is administrationthrough various routes, including, subcutaneous, intravenous,intradermal, intramuscular, intraperitoneal, intranasal, transdermal, orbuccal routes. Subcutaneous administration is preferred. Parenteraladministration are achieved, for example, by bolus injection or bygradual perfusion over time.

A typical regimen for preventing, suppressing, or treating a disease orcondition which can be alleviated by a cellular immune response byactive specific cellular immunotherapy, comprises administration of aneffective amount of the composition as described above, administered asa single treatment, or repeated as enhancing or booster dosages, over aperiod up to and including one week to about 48 months.

According to the present invention, an “effective amount” of acomposition is an amount sufficient to achieve a desired biologicaleffect, in this case at least one of cellular or humoral immune responseto a papillomavirus genotype. It is understood that the effective dosagewill be dependent upon the age, sex, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired. The most preferred dosage will betailored to the individual subject, as is understood and determinable byone of skill in the art, without undue experimentation.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims. The contents of all references, patents and published patentapplications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1

Establishment of Serum-free SF-9 Insect Cell Line

A new insect cell line designated Sf-9S was derived from the parent S.frugiperda Sf-9 cell line (ATCC CRL-1771) by several rounds of selectiveprocesses based on serum-independent growth and enhanced expression ofsecreted recombinant proteins from baculovirus vectors. Specifically,Sf-9 cells were cultivated to passage 38 in Grace's insect media (LifeTechnologies, Grand Island, N.Y. 14072) supplemented with 10% fetalbovine serum (Life Technologies, Grand Island, N.Y. 14072) as monolayercultures in T-75 flasks (Corning, Inc., Corning, N.Y.). The master cellbank of Sf-9 cells was stored at passage 38 in serum-containing media at−70° C. and in liquid nitrogen. A working cell bank was established froma single cryovial of the Sf-9 master cell bank and cultivated inserum-containing insect media for an additional five (5) passages.

Initially, cell clones capable of growing in commercial serum-free mediaas suspension cultures were isolated from monolayer cultures of parentSf-9 cells dependent on serum-containing media by sequential weaning ofparent cells from serum-containing media This process involved theplating of cell aliquots (200 μl) from a cell suspension (one cell per200 μl) of the parent cell line in serum-containing media onto 96-welldishes at a ratio of 200 μl per well. Following attachment of cells andinspection of wells for wells with more than one cell, the media waschanged from serum-containing media (100%) to a media mixture comprisedof 75% serurn-containing media and 25% serum-free media. After one totwo weeks in culture, the media was changed from wells that initiallycontained only one cell per well and demonstrated cell growth andreplication (i.e. four to five cells).

The second media mixture was comprised of 50% serum-containing media and50% serum-free media. The cells were allowed to grow for another one totwo weeks. The media was changed from wells containing cells thatcontinued to grow and replicate. The third media mixture was comprisedof 25% serum-containing media and 75% serum-free media The cells wereallowed to grow and replicate. After another two to four weeks, themedia was changed from wells containing cells that continued to grow andreplicate. The final media was comprised of serum-free media (100%).During each round of the weaning process, more than 90% of the cells didnot survive the reduction in serum. This high level of cell deathcreated a selective pressure to permit development of a new cellphenotype. Cells from wells that demonstrated continuous cell growth andreplication were harvested and seeded into larger culture vessels. Whena total cell density of >4×10⁶ cells was obtained, cells were seededinto shaker flasks (50 ml) as 10 ml suspension cultures with a startingcell density of 0.2-0.5×10⁶ cells/ml. Eight (8) clones that grewexponentially to a saturation cell density of >6×10⁶ cells/ml inserum-free media was selected, expanded, and frozen. One of the cloneswas established as a serum-free independent cell line.

Example 2

Establishment of Transformed SF-9S Cell Line

In a second selection process, one of the serum-free cell clonesdeveloped in Example 1 was chosen to select cell clones that may produceenhanced levels of recombinant extracellular proteins and VLPs fromseveral viruses including rotaviruses and human papillomaviruses bysuccessive rounds of clonal selection of cells infected with recombinantbaculoviruses and expressing extracellular self-assembled VLPs.

This process involved the plating of cell aliquots (200 μl) from a cellsuspension (one cell per 200 μl) of the parent cell clone (#23) inserum-free media onto 96-well dishes at a ratio of 200 μl per well. Fromwells containing a single cell in the original seeding, cells were grownto confluency and subcultured into six replica-plates (96-well). Thefirst round of selection was performed when a total cell density of2-4×10³ cells/well was obtained; the cells were infected withrecombinant baculoviruses encoding human rotavirus virus VP2 and VP6capsid genes. After three days of baculovirus infection, the infectedcells and extracellular media were harvested by centrifugation. Infectedcells were solubilized by adding 250 μl of 1% sodium dodecyl sulfate(SDS) and 10 mM β-mercaptoethanol (β-ME). SDS and β-ME were added toextracellular supernatants to final concentrations of 1% and 10 mM,respectively. Aliquots (10 μl) of solubilized cell lysates andextracellular media were heat-denatured (99° C. for 10 min.) underreduced conditions and analyzed by SDS-PAGE and Western blotting usingantisera to rotaviruses.

After review of the test results from the first virus infection, twentyfour (24) cell clones demonstrating the highest levels of extracellularrecombinant rotavirus VLPs were identified, seeded into 96-well plates,grown to confluency, and infected with a second recombinant baculovirusencoding HPV-16 L1 capsid protein. At three days post-infection,infected cells and extracellular supernatants were produced bycentrigation of infected cell suspensions from the plate.

Infected cells and extracellular supernatants were analyzed by SDS-PAGEand Western blot analyses using polyclonal anti-HPV-16 L1 sera. The testresults of both viral infections were reviewed and compared. One cellclone (#12) that produced the high levels of extracellular VLPs fromrotavirus and HPV capsid proteins was chosen to establish a cell linecapable of producing extracellular VLPs. To establish a cell line fromthe selected cells, cells from an uninfected replica plate wereamplified at 28° C. and 150 rpm in a platform shaker incubator into asuspension culture using Sf-900 II serum free media (GIBCO). Theamplified cell culture was diluted to a seeding cell density of 0.25×10⁶cells/ml, grown in 100 ml of Sf-900 II SFM within a 500 ml shaker flask,and subcultured at a split ratio of 1:20 for forty three passages. Aftercontinuous passaging, the cell line was established and was passagedthree more times to establish a master cell bank.

Example 3

Cloning Codon-optimized HPV-16 L1 Genes and Establishment of RecombinantBaculovirus Stocks

A BPV-16 L1 prototype (GenBank Accession No. K02718) and modified inU.S. Pat. No. 5,985,610, was optimized for codon usage in insect cellsof the Lepidopteran family. The HPV-16 L1 gene was optimized (FIG. 1A)in this embodiment of the present invention for codon usage based on thefollowing criteria: (1) abundance of aminoacyl-tRNAs for a particularcodon in Lepidopteran species of insect cells for a given amino acid asdescribed by Levin and Whittome (2000); (2) maintenance of GC-AT ratioin L1 gene sequence at approximately 1:1; (3) minimal introduction ofpalindromic or stem-loop DNA structures, and (4) minimal introduction oftranscription and post-transcription repressor element sequences.

The optimized gene sequence was synthesized in vitro as overlappingoligonucleotides, cloned into a subcloning plasmid vector, and thencloned into a bacmid transfer vector (i.e., Luckow et al., 1993),according to procedures known in the art (i.e., Summers and Smith,1987). The bacmid transfer vector pFASCTBAC1 with HPV-16 L1 gene wasused to transform competent E. coli DH10BAC cells and producerecombinant bacmid DNA. The recombinant bacmid DNA with the L1 gene wastransfected into insect cells to produce recombinant baculovirusesencoding L1 genes.

In particular, a restriction fragment (Bam HI/Sal I restriction fragment(1572 bp) containing a HPV-16 L1 gene (K strain)) containing a HPV L1capsid gene from a natural virus isolate or synthesized gene is ligatedto a bacmid transfer vector, such as pFASTBAC-1 (see, for example,Luckow et al., 1993), at the multiple cloning site, which contains a Tn7transposable element surrounded by the transcription promoter andpolyadenylation/transcription termination elements of the polyhedrin(polh) gene from a wild type AcMNPV genome. Competent E. coli DH10BACcells, which contain bacmid DNA (an AcMNMV baculovirus genome with a Tn7transposable element within the polyhedrin locus), are transformed withthe bacmid transfer vector containing the HPV L1 gene.

Recombinant bacmids are produced by site-directed recombination betweenthe respective Tn7 transposable elements of the transfer vector and thebacmid genome resulting in the production of recombinant bacmid genomesencoding the optimized L1 gene in the E. coli hosts. The recombinantbacmid DNA is isolated for example by miniprep DNA isolation andtransfected into Sf-9S insect cells to produce recombinant baculovirusesencoding the L1 genes.

The progeny recombinant baculoviruses (˜10⁴ plaque forming units) areplaque-purified (3×) and selected for high expression of the HPV-16 L1gene product, as determined by SDS-PAGE and Western blot analyses usingrabbit polyclonal antisera specific for the HPV 16 L1 gene product(Pharmingen). A HPV-16 L1 master virus stock is prepared in Sf-9S insectcells, as described in Example 2, from one of the plaque-purified clonesexpressing high levels of recombinant HPV-16 L1 proteins thatself-assemble into virus-like particles and is qualified for safety andbiological properties as described below. Working virus stocks of HPV-16L1-expressing baculoviruses are prepared by infection of Sf-9S insectcells at a multiplicity of infection of 0.1 pfu/cell with the qualifiedHPV-16 L1 master virus stock and are characterized as described below toqualify for recombinant HPV-16 L1 VLP product manufacturing.

Example 4

Cloning Codon-optimized HPV-16 Chimeric Genes and Establishment ofChimeric Recombinant Baculovirus Stocks

HPV-16 L2 fusion genes are optimized for codon usage in insect cells asdescribed above for L1 genes (FIG. 1C-1E). The L2/E7/E2 fusion genesequence is synthesized in vitro as overlapping oligonucleotides, clonedinto a subcloning plasmid vector, and then cloned into a bacmid transfervector according to procedures known in the art (i.e., Luckow et al.,1993; Summers and Smith, 1987).

For example, a baculovirus transfer vector with a L2 fusion gene isco-transfected into insect cells with linearized wild-type baculovirusgenomic DNA to produce recombinant baculoviruses encoding L2 fusiongenes. Alternatively, the bacmid transfer vector with the L2 fusion geneis used to transform competent E. coli DH10BAC cells and producerecombinant bacmid DNA. The codon-optimized gene is cloned into a bacmidtransfer vector (i. e., Luckow et al., 1993).

In particular, a Bam HI/Kpn I restriction DNA fragment (2834 bp)containing a HPV-16 L2/E7/E2 fusion gene was ligated with T4 DNA ligaseto a Bam HI/Kpn I digest of the bacmid transfer vector pFASTBAC-1(Luckow et al., 1993) at the multiple cloning site. Competent E. coliDH10BAC cells were transformed with the bacmid transfer vectorcontaining the HPV-16 L2/E7/E2 fusion gene. Recombinant bacmids wereproduced by site-directed recombination between the respective Tn7transposable elements of the transfer vector and the bacmid genomeresulting in the production of recombinant bacmid genomes encoding theoptimized HPV L1 gene in the E. coli hosts.

The recombinant bacmid DNA was isolated by miniprep DNA isolation andtransfected into Sf-9S insect cells to produce recombinant baculovirusesencoding the HPV-16 L2/E7/E2 L2 fusion genes. The recombinantbaculoviruses were plaque-purified (3×) in Sf-9S insect cells andselected for high expression of the HPV-16 L2, E7, and E2 gene products,as determined by SDS-PAGE and Western blot analyses using antiseraspecific for each peptide within the L2 fusion gene product (HPV-16 L2,E7, and E2 peptides). A master virus stock of baculoviruses expressingHPV-16 L2/E7/E2 fusion proteins was prepared in Sf-9S insect cells fromone of the plaque-purified clones expressing high levels of recombinantHPV L2 fusion proteins and was qualified for safety and biologicalproperties. Working virus stocks of baculoviruses expressing HPV-16L2/E7/E2 fusion proteins were prepared in Sf-9S insect cells at amultiplicity of infection of 0.1 pfu/cell with the qualified mastervirus stock and were tested.

Example 5

Characterization and Qualification of L1 and Chimeric RecombinantBaculovirus Stocks

Recombinant baculovirus stocks for each of the HPV L1 and L2 fusionviruses were established. Master and working virus stocks wereestablished from high expression virus clones and characterized forsafety and biological properties. Safety properties of master andworking virus stocks included microbial sterility, adventitious agentpresence, endotoxin level, spiroplasma, and mycoplasma contaminants, andthe like. Biological properties included genetic identity, virus titer,viral replication competence, and recombinant protein productioncompetence. The genetic identity of the master virus stock wasdetermined, for example, by DNA sequence analysis of both strands ofbacmid DNA encoding the HPV L1 or L2 fusion genes and flankingsequences. The virus titer of master and working virus stocks weredetermined by an agarose plaque assay using insect cells and serialdilutions of the virus stock.

Viral replication competency was evaluated by passage of an aliquot ofthe virus stocks in insect cells at low multiplicity of infection.Subsequent determination of the virus titer for the progeny viruspassage was performed by agarose plaque assay. Recombinant proteinexpression competency was evaluated, for example, by infection of insectcells with an aliquot of the virus stocks and subsequent determinationof the relative abundance of recombinant proteins such HPV L1 and L2fusion proteins per total cell protein in infected cells by SDS-PAGEanalysis.

Example 6

Virus Infection for HPV-16 L1 VLPS

Recombinant HPV-16 L1 VLPs expressed in baculovirus-infected Sf-9S cellswere purified from intracellular and extracellular crude lysates. Sf-9Sinsect cells from Example 2 were thawed from a single cryovial of theworking cell bank frozen at −70° C. in Sf-900 II SFM insect cell media(GIBCO) at a concentration of 1.0×10⁷ cells/ml. Thawed cells were seededinto 50 ml of Sf-900 II SFM insect cell media and cultured as suspensioncultures in 500 ml shaker flasks in a platform shaker incubator at 28°C. with an agitation speed of 125 rpm.

After the cell density reached 6×10⁶ cells/ml and a cell viability ofmore than 95%, the culture was seeded into two (2) liter flasks in afinal volume of 800 ml of insect serum-free media per flask at astarting seed density of 0.5×10⁶ cells/ml. The cells were cultured in aplatform shaker incubator at 28° C. with an agitation speed of 100-125rpm. When the cell density reached 2-3×10⁶ cells/ml, the insect cellswere infected with a recombinant baculovirus encoding the HPV-16 L1capsid gene (K strain) from the polh locus made according to Example 3.The virus infection was established at a MOI of 3 pfu per cell. Thevirus infection was carried out for six days in a platform shakerincubator at 28° C. with an agitation speed of 125 rpm. The infectedcells were harvested by centrifugation (at 1,500×g and 2-8° C. for 10minutes) after the following conditions were met: cell viability wasless than 25%, and L1 gene products were in culture fluids and withininfected cells.

Example 7

Virus Infection for HPV-16 Chimeric VLPS

Production of intracellular HPV-16 L2/E7/E2 chimeric VLPs began withhigh multiplicity infection of log phase Sf-9S insect cells (1.5×10⁶cells/ml) in suspension shaker flask cultures (2 L) containingserum-free insect cell medium (800 ml; HyQ SFM media, HyClone) madeaccording to Example 2 with aliquots of HPV-16 L1 and L2/E7/E2 workingvirus stocks prepared according to Example 4. The ratio of co-infectingviruses was approximately 1:10 (L1 to L2 virus). The virus infection wasmonitored daily by the trypan blue exclusion method for cytopathiceffects, cell viability, and cell density and by SDS-PAGE and Westernblot analyses of recombinant HPV L1 and L2 fusion proteins in infectedcells. At three days post-infection when peak recombinant HPV-16 L1 andL2/E7/E2 fusion gene expression occurred, infected cells containingintracellular HPV-16 chimeric VLPs were harvested by centrifugation at1500×g and 2-8° C. for 5 minutes and processed to obtain purified HPV-16chimeric VLP products.

Example 8

Preparation of Crude Cell Lysates for HPV-16 Chimeric VLPs

Recombinant HPV-16 chimeric VLPs including recombinant HPV-16 L2/E7/E2fusion proteins encapsulated into HPV-16 L1 VLPs produced according toExample 6 were harvested from modified Sf-9 insect cells infected withrecombinant baculoviruses encoding the HPV-16 L1 capsid gene (K strain)and HPV-16 L2/E7/E2 fusion gene. Infected cells were harvested bylow-speed centrifugation at 1,500×g and 2-8° C. for 10 minutes. Infectedcell pellets containing intracellular recombinant HPV-16 chimeric VLPswere resuspended in phosphate-buffered saline II solution (1.54 mMKH₂PO₄, 2.71 mM Na₂HPO₄.7H₂O, and 154 mM NaCl (pH 7.2)) at a ratio of 10ml buffer per gram of cell pellet. Protease inhibitors corresponding toserine, cysteine, and aspartate classes of proteases were added to thefollowing final concentrations: (PMSF, 1 mM; Aprotinin, 1 μg/ml;Leupeptin, 10 μg/ml; Pepstatin, 5 μg/ml). The resuspended cellscontaining intracellular recombinant papillomavirus VLPs were disruptedby mild sonication in phosphate-buffered saline solution with two (2)pulses at 200-300 watts and 2-8° C. with a Branson Model 250 sonifierequipped with a ⅛″ probe. The result of sonication was a crude celllysate containing intact intracellular recombinant HPV-16 chimeric VLPswith minimal disruption of cellular proteosomes and degradation ofHPV-16 L2/E7/E2 fusion proteins.

Example 9

Clarification of HPV-16 L1 VLP Crude Cell Lysates and Supernantants

Crude cell lysates made according to Example 7 containing intracellularrecombinant HPV-16 L1 VLPs were clarified by centrifugation at 12,000×gand 2-8° C. for 60 minutes to remove large cellular debris andmembranes. Clarified supernatants were collected by aspiration, andcellular pellets were discarded. Crude media supernatants containingextracellular recombinant HPV-16 L1 VLPs were clarified bycentrifugation at 12,000×g and 2-8° C. for 60 minutes to remove largecellular debris and membranes. Clarified supernatants were collected byaspiration, and cellular pellets were discarded.

Example 10

Clarification of HPV-16 Chimeric VLP Crude Cell Lysates

Crude cell lysates made according to Example 8 were purified as follows.The cell lysate containing intracellular recombinant HPV-16 chimericVLPs harvested from insect cells infected with recombinant baculovirusesencoding HPV-16 L1 and L2/E7/E2 fusion proteins were clarified bycentrifugation at 12,000×g and 2-8° C. for 60 minutes to remove largecellular debris and membranes. Clarified supernatants were collected byaspiration, and cellular pellets were discarded.

Example 11

Concentration and Diafiltration of HPV-16 VLP Clarified Supernatants

Concentration and diafiltration steps of the present invention involvedultrafiltration of clarified supernatants made according to Example 9.The clarified supernatant contained intracellular and extracellularrecombinant HPV-16 L1 VLPs. These VLPs were expressed from infection ofSf-9S insect cells with recombinant baculovirus encoding the L1 capsidgene of HPV-16 (K strain). Clarified supernatants containingintracellular and extracellular recombinant HPV-16 L1 VLPs wereconcentrated ten fold by ultrafiltration using an Amicon M-12 ProfluxTangential Flow Ultrafiltration System equipped with a hollow fiberultrafiltration cartridge (AIG Technologies Model UFP-500-C-55A).Concentrates containing intracellular and extracellular recombinantBPV-16 L1 VLPs were diafiltered against eight volumes of cation exchangeloading buffer solution containing 20 mM sodium phosphate (pH 5.7) and10 mM sodium chloride by ultrafiltration using an Amicon M-12 ProfluxTangential Flow Ultrafiltration System equipped with a hollow fiberultrafiltration cartridge (A/G Technologies Model UFP-500-C-55A) at aninitial flow rate of 0.8 L/min. and an inlet/outlet pressure of 8 psi.

Example 12

Cation Exchange Chromatography of HPV-16 VLP Concentrated Dialysates

Diafiltered concentrates containing intracellular and extracellularrecombinant HPV-16 VLPs made according to Example I1 were loaded onto achromatography column containing Streamline SP Adsorptive resin(Amersham Biosciences), a strong cation exchange chromatography resin,at a flow rate of 0.5 L/hr and a ratio of 1 ml resin per 1 gram ofdiafiltrate. The chromatographic column was developed with a Waters 6000HPLC System. The SP resin was equilibrated with a cation binding buffer(50 mM sodium phosphate (pH 5.7) and 10 mM sodium chloride). Followingbinding of diafiltrates containing intracellular and extracellularrecombinant HPV-16 VLPs, the column was rinsed with five (5) volumes ofcation binding buffer. Bound proteins were eluted as 1 ml fractions withUV-monitoring at 214 nm from the column resin at a flow rate of 0.5 L/hrusing a step pH gradient from 6.0 to 8.0 in 20 mM sodium phosphate with10 mM sodium chloride. Fractions containing intracellular andextracellular recombinant HPV-16 VLPs eluted in the 7.0-7.5 steps, asdetermined by SDS-PAGE and Western blot analysis of elution fractionsamples using antisera against papillomavirus L1 capsid proteins. Thosefractions containing HPV-16 L1 VLPs were pooled.

Example 13

Affinity Chromatography of HPV-16 VLP Cation Exchange ChromatographyEluates

Pooled eluates from the cation exchange chromatography that containintracellular and extracellular recombinant HPV-16 L1 VLPs madeaccording to Example 12 were dialyzed against 100 volumes of affinityloading buffer (20 mM sodium phosphate (pH 5.7), 2 mM EGTA, and 300 mMsodium chloride) for 8-16 hrs. The dialyzed material was loaded onto acolumn containing heparin agarose (Amersham Biosciences) at a flow rateof 1 ml/min. The ratio of packed heparin agarose to protein was 1 ml ofresin per 0.5 grams of protein. Bound proteins including recombinantHPV-16 L1 VLPs were eluted as 1 ml fractions with UV-monitoring at 214nrn from the column resin at a flow rate of 1 ml/min. using a linearsalt gradient from 300 mM to 2 M. Fractions containing recombinantHPV-16 L1 VLPs eluted in salt fractions from 500-700 mM, as determinedby SDS-PAGE and Western blot analysis of elution fraction samples usingantisera against papillomavirus L1 capsid proteins. Those fractionscontaining HPV-16 L1 VLPs were pooled.

Example 14

Alternative Affinity Chromatography of HPV-16 VLP Cation ExchangeChromatography Eluates

Pooled eluates from cation exchange chromatography that containintracellular and extracellular recombinant HPV-16 L1 VLPs madeaccording to Example 12 were dialyzed against 100 volumes of affinityloading buffer (20 mM sodium phosphate (pH 5.7), 2 mM EGTA, and 300 mMsodium chloride) for 8-16 hrs. The dialyzed material was loaded onto acolumn containing Matrex Cellufine Sulfate (Amersham Biosciences) at aflow rate of 1 ml/min. The ratio of packed Matrex Cellufine Sulfate toprotein was 1 ml of resin per 0.5 grams of protein. Bound proteinsincluding recombinant HPV-16 L1 VLPs were eluted as 1 ml fractions withUV-monitoring at 214 nm from the column resin at a flow rate of 1 ml/minusing a linear salt gradient from 300 mM to 2 M. Fractions containingrecombinant HPV-16 L1 VLPs eluted in salt fractions from 400 mM to 600mM, as determined by SDS-PAGE and Western blot analyses of elutionfraction samples using antisera against papillomavirus L1 capsidproteins. Those fractions containing HPV-16 L1 VLPs were pooled.

Example 15

Anion Displacement Chromatography of HPV-16 VLP Affinity ChromatographyEluates

Pooled eluates from affinity chromatography that contain recombinantHPV-16 L1 VLPs made according to Examples 13 or 14 were dialyzed against100 volumes of anion loading buffer (0.24 M Tris-HCl (pH 8.0)) for 8-16hrs. The dialyzed material was loaded onto a column containing QSepharose FF (Amersham Biosciences), a strong anion exchangechromatography resin, at a flow rate of 0.5 ml/min. The ratio of packedQ Sepharose to protein was 1 ml of resin per 0.1 gram of protein. Boundproteins including recombinant papillomavirus VLPs were displaced as 1ml fractions with UV-monitoring at 214 rM from the column resin at aflow rate of 0.5 ml/min using a linear gradient from 0 to 5 mg/midextran sulfate (5000 MW). Fractions containing recombinant HPV-16 L1VLPs eluted in dextran sulfate fractions from 4 mg/ml to 5 mg/ml, asdetermined by SDS-PAGE and Western blot analyses of elution fractionsamples using antisera against papillomavirus L1 capsid proteins. Thosefractions containing HPV-16 L1 VLPs were pooled. Pooled eluates fromanion exchange chromatography that contain recombinant papillomavirusVLPs were dialyzed against 100-150 volumes of final bulk storage buffer(5 mM Na₂HPO₄.7H₂O, 5 mM KH₂PO₄, and 500 mM NaCl (pH 6.8)) for 8-16 hrs.

Example 16

Linear Sucrose Gradient Purification as an Alternative To L1 VLPChromatographic VLP Purification

Intracellular and extracellular HPV VLPs were also purified fromconcentrated crude cell lysates and media supernatants made according toExamples 9 or 10 by ultracentrifugation on linear sucrose gradients.Concentrates (5-10 g) containing HPV L1 VLPs were loaded at a flow rateof 100-250 ml per minute onto 0-65% linear sucrose gradient prepared inphosphate-buffered saline solution (5 mM potassium phosphate(monobasic), 5 mM sodium phosphate (dibasic), 154 mM sodium chloride (pH7.2)) in a RK-2 vertical rotor (1.6 L) accelerating at 35,000 rpm in aRK continuous flow ultracentrifuge (Schiaparelli Instruments, Amsterdam,The Netherlands). The gradient was resolved by centrifugation at 35,000rpm and 15-25° C. for one to two hours residence and one hour coastingto a complete stop without braking. Gradient material from the sucrosegradient was passed through a ULV_(218nm) monitor and collected as 50 mlaliquots in a fraction collector. Samples from each fraction weresubjected to SDS-PAGE and Western blot analyses to find HPV-16 L1 VLPs.Results from these analyses indicated that recombinant HPV-16 L1 VLPssedimented into two bands corresponding to 43-53% sucrose and 30-40%sucrose. The baculoviruses sedimented as one band corresponding to30-35% sucrose. Fractions 6-9 containing recombinant HPV-16 L1 VLPscomprised of HPV-16 L1 protein species with molecular weights of 55 and60 kD were pooled. Fractions 10-14, which contained recombinant HPV-16L1 proteins with molecular weights of 50 and 55 kD and proteolyticbreakdown products of L1 proteins, were not pooled and used as productdue to the proteolysis.. The pooled L1 VLP fractions were diluted 6 foldwith the PBS solution, and subjected to a second round ofultracentrifugation on linear sucrose gradients, except the secondgradient was 0-50% sucrose (PBS) and was run for a total of two hours(one hour residence and one hour coasting). Sucrose gradient fractionsfrom 0-50% linear sucrose gradients were examined for HPV-16 L1 VLPs bySDS-PAGE, SEC HPLC, Western blot, and ELISA analyses. Fractionscontaining intact HPV VLPs displaying conformational epitopes werepooled.

Pooled sucrose gradient fractions containing extracellular recombinantHPV-16 L1 VLPs were diafiltered by ultrafiltration against eight volumesof final VLP storage buffer containing 5 mM Na₂HPO₄.7H₂O, 5 mM KH₂PO₄,and 500 mM NaCl (pH 6.8) using an Amicon M-12 Proflux Tangential FlowUltrafiltration System equipped with a hollow fiber ultrafiltrationcartridge (A/G Technologies Model UFP-500-C-55A) at an initial flow rateof 0.8 L/min and an inlet/outlet pressure of 8 psi. Triton X-100, anonionic detergent and surfactant, was added at a final concentration of0.1% to the bulk HPV VLP product, which was incubated for two hours atambient temperature to inactivate residual baculoviruses. Alternatively,the bulk HPV VLP product was irradiated with at least three rounds ofultraviolet (UV) light at 254 nm to inactivate residual baculoviruses.The treated material was filtered aseptically through a 0.22 μm membraneinto silanized borosilicate glass containers.

Example 17

Sucrose Step Gradient Purification of HPV-16 L1 VLPS as an Alternativeto L1 VLP Chromatographic VLP Purification

In yet another alternate embodiment of the present invention,diafiltered concentrates made according to Examples 9 and containingHPV-16 L1VLPs were purified by rate-zonal ultracentrifugation ondiscontinuous sucrose step gradients. Diafiltrates containingrecombinant HPV-16 L1VLPs were loaded onto approximately 25% sucrosecushions (prepared in PBS solution) in a swinging bucket rotor (SorvalModel AH 628) accelerating at 35,000 rpm and 2-8° C. in anultracentrifuge (Sorval Model OTB-65B) for three hours. The pellets atthe bottom of the sucrose cushion were collected, while the sucrosecushion and load material were discarded. The sucrose cushion pelletswere solubilized in PBS solution at approximately 1 g of pellet per mlof buffer 1 and loaded onto sucrose step gradients containing six stepscomprising approximately 25, 30, 35, 40, 45, 50 and 65% sucrose. Thesucrose step gradients were resolved by ultracentrifugation in aswinging bucket rotor at 35,000 and 2-8° C. for 1 hour until recombinantHPV VLPs separate from baculovirus particles.

Gradient material from the first round of sucrose step gradients weremonitored by ultraviolet light during collection in a fractioncollector. Gradient fractions were analyzed by SDS-PAGE-and Western blotanalysis using antisera against HPV-16 L1 capsid proteins. Peakfractions containing HPV VLPs or their component proteins were pooled,diluted multifold with buffer solution, and were subjected to a secondround of ultracentrifugation on sucrose step sucrose gradients. Gradientmaterial from the second round of sucrose step gradients were monitoredby ultraviolet light during collection in a fraction collector. Gradientfractions from the second round of ultracentrifugation were analyzed bySDS-PAGE and Western blot analysis using antisera against papillomavirusL1 capsid proteins. Peak fractions containing HPV-16 proteins werepooled. The purified recombinant HPV-16 L1 VLPs in the pooled fractionswere formulated as recombinant HPV-16 L1 VLP products.

Example 18

Sucrose Step Gradient Purification of HPV-16 Chimeric L1 VLPs

In yet another alternate embodiment of the present invention,diafiltered concentrates made according to Example 10 and containingHPV-16 chimeric VLPs were purified by rate-zonal ultracentrifugation ondiscontinuous sucrose step gradients based primarily on mass ratherdensity of recombinant HPV-16 chimeric VLPs in sucrose.

Diafiltrates containing recombinant HPV-16 chimeric VLPs were loadedonto approximately 25% sucrose cushions in a swinging bucket rotoraccelerating at 35,000 rpm in an ultracentrifuge (Sorval) for threehours. The pellets at the bottom of the sucrose cushion were collected,while the sucrose cushion and load material were discarded. The sucrosecushion pellets were solubilized in PBS buffer and loaded onto sucrosestep gradients containing six steps comprising approximately 25, 30, 35,40, 45, 50 and 65% sucrose.

The sucrose step gradients were resolved by ultracentrifugation in aswinging bucket rotor at 35,000 for 1 hour until recombinant HPV VLPswere separated from baculovirus particles. Gradient material from thefirst round of sucrose step gradients was monitored by ultraviolet lightduring collection in a fraction collector. Gradient fractions wereanalyzed by SDS-PAGE and Western blot analysis using antisera againstHPV-16 L1 capsid proteins and L2, E7, and E2 fusion proteins. Peakfractions containing HPV VLPs or their component proteins were pooled,diluted multifold with buffer solution, and were subjected to a secondround of ultracentrifugation on sucrose step sucrose gradients.

Gradient material from the second round of sucrose step gradients weremonitored by ultraviolet light during collection in a fractioncollector. Gradient fractions from the second round ofultracentrifugation were analyzed by SDS-PAGE and Western blot analysisusing antisera against papillomavirus L1 capsid proteins and/or L2fusion proteins. Peak fractions containing HPV-16 proteins were pooled.The purified recombinant HPV-16 chimeric VLPs in the pooled fractionswere formulated as recombinant HPV-16 chimeric VLP products.

Example 19

Formulation of Final Bulk HPV-16 L1 VLP Products

Residual baculovirus present in recombinant HPV-16 L1 VLPs madeaccording to Examples 15, 16, or 17 was inactivated by treatment ofcrude, intermediate, and final bulk products with the non-ionicdetergent and surfactant, Triton X-100. Inactivation of virus in HPV-16L1 VLP products was provided by addition of Triton X-100 at a finalconcentration 0.1% for 2 hours at 15-25° C. Following treatment, HPV VLPproducts were diaflitered against 4×1000 volumes of high salt buffercontaining (5 mM Na₂HPO₄.7H₂O, 5 mM KH₂PO₄, an 500 mM NaCl (pH 6.8)) and2-8° C. for 12 hours. Diafiltrates containing treated recombinant HPV-16L1 VLPs were filtered aseptically through a 0.22 μm membrane at 15-25°C. Filtered HPV-16 L1 VLP final bulk products were dispensed into tenliter containers including 316L stainless steel tanks, silanizedborosilicate glass bottles, and polyethylene plastic bioprocess bags andstored at 2-8° C. for <six (6) months or at <−70° C. for <2 years.

Example 20

Analysis of Intact HPV VLPS

ELISA testing of solutions containing recombinant HPV-16 L1 VLPs (lot1274), sucrose, and/or clarified supernatants from bHPV-16 L1 infectedSf-9S insect cells mock treated or treated two hours at room temperaturewith Triton X-100 (0.1% final concentration) was performed. Mock andtreated solutions containing recombinant HPV-16 L1 VLPs were dialyzedagainst four changes of 1000 volumes of phosphate buffer containing 0.5M sodium chloride. Various amounts (250, 500, and 1000 ng) of dialysateswere bound to 96-well plates and reacted with antibodies from the murinehybridoma cell line H16.V5 that bind to HPV-16 L1 protein conformationalepitopes that elicit neutralizing antibodies to determine the effect ofTriton X-100 on these epitopes. The results as shown in FIG. 11demonstrated that Triton had little or no effect on recombinant HPV-16L1 VLPs with or without sucrose, but five fold decrease in bindingactivity was noted with 1000 ng of VLPs mixed with supernatants ascompared to that of mock treated and Triton-treated VLPs or VLPs insucrose. These results indicated that Triton treatment of recombinantVLPs, which effectively afforded as much as 4 to 7 log reduction inbaculovirus titers, did not irreversibly destroy conformational L1epitopes of recombinant HPV-16 L1 VLPs in buffer or buffer with sucrose.However, VLPs amidst infected extracellular materials did not retain theproper epitope conformation after Triton treatment. The conclusion: thebest time to add Triton in the purification of recombinant BPV VLPs wasafter downstream processing but prior to terminal filtration of finalbulk products.

In one set of analyses as depicted in FIG. 12, samples (5 μg ofphosphate buffered saline) from different lots (1207, 1244, 1265, and1268) of recombinant HPV-16 L1 VLPs were injected to an analytical sizeexclusion column and resolved according to their mass and shape. Thepre-poured column used for analytical size exclusion chromatographicanalysis was a 15 cm TSK 6000PWX stainless steel column (Tosohaus). Thefractionation range of this column was more than 1,000,000 to 20,000daltons. The volume of test sample injected into the column was 50 μl. AWaters 6000 BPLC system using Millennium computer software provided theautomated mechanics and programs necessary for sample injection, buffertransfer, column development, UV monitoring, fraction collection, andprotein data management.

As the result of the analysis, control blue dextran beads having amolecular weight of 2,000,000 daltons was resolved as a single peak at10-12 minutes. The expected resolution of VLPs was at 15-16 minutes andmonomeric proteins at 24-26 minutes. Pentameric HPV-16 L1 structureswere expected to resolve at 19-20 minutes when present. Analysis of afinal container vaccine vial production lot 1207 stored at −70° C. formore than two years indicated that the recombinant HPV-16 L1 VLPsresolved as two peaks (FIG. 12). The major peak was at 15 min. and aminor peak was at 25 min. Upon integration of the area beneath eachpeak, these data indicated that at least 95% of the L1 protein in thisproduction remained as VLPs after more than two years frozen at −70° C.Results of samples from bulk lots 1244, 11265, and 1268 of recombinantHPV-16 L1 VLP showed in FIG. 12 a single major peak at 15 min. Followingintegration of the peak areas, >95% of this vaccine product remained asVLPs more than two years after production. As recombinant HPV-16 L1 VLPsdissociate, pentamers at 20 min and monomers at 25 min. appeared (datanot shown). Interestingly, bovine papillomavirus (BPV) VLPs, a relativeof HPV-16, behaved similarly in this assay. Hawaii virus (HV) VLPs,calicivirus self-assembled macromolecules, exhibited >99% intact VLPs.

An electron micrograph of HPV VLPs was obtained by negative staining of10 μl aliquots of recombinant HPV-16 L1 VLPs (1 mg/ml in phosphatebuffered saline) with uranyl acetate and observing at high magnification(36,000×) on a transmission electron microscope (Phillips). Theseresults as depicted in FIG. 13 demonstrated intact and discretevirus-like particles with a size of 40 to 55 nm and a morphology similarto papillomavirus virions. These electron micrographs provided anotherexample of confirmatory evidence of high quality HPV-16 L1. Further, theSEC BPLC assay was shown again to be an effective measurement tool forquantitation of VLPs.

Example 21

Production of Antisera to Host Contaminant Proteins

Antisera to insect cell and wild type baculovirus proteins were producedto detect contaminating host proteins in baculovirus-derived recombinantprotein products. For antiserum against insect cell proteins, 800 mlsuspension shaker cultures of Sf-9S and High Five insect cells weregrown to cell density of 2×10⁶ cell per ml. The cultured cells wereharvested by centrifugation at 500×g and 2-8° C. for five minutes. Thecell pellets were resuspended in 10 ml of phosphate-buffered salinesolution (5 mM sodium phosphate, dibasic, 5 mM potassium phosphate,monobasic, 154 mM sodium chloride (pH 7.2)). The resuspended cells weredisrupted by sonication with two (2) pulses at 200-300 watts and 2-8° C.with a Branson Model 250 sonifier equipped with a ⅛″ probe to producecell lysates. The sonicated cell lysates were clarified bycentrifugation at 12,000×g and 2-8° C. for 60 minutes to remove largecellular debris and membranes. The clarified supernatants were retainedfor protein quantitation by the BCA method, and the pellets werediscarded.

Aliquots of the clarified cell lysates were formulated into immunogensby emulsification of equal volumes of antigen and complete Freund'sadjuvant (DIFCO) at an antigen concentration of 200 μg/ml. Theimmunogens were administered intramuscularly (primary) as two doses (50μg/dose) into the hindquarters of New Zealand rabbits. At four weekspost-immunization, a second round of immunization (booster) occurred asbefore except that incomplete Freund's adjuvant was used. At eight weekspost-immunization, sera were isolated from blood withdrawn fromimmunized animals.

The antibody titers of the immunized sera were determined by Westernblot analysis using nitrocellulose membranes containing proteins fromSf-9S and High Five insect cells, as well as control protein samplesfrom baculovirus- and E. coli-derived recombinant protein products.Results using these antisera from Sf-9S insect cell proteinsdemonstrated that the antibodies specific for this cell line werepresent, as positive binding was observed in lanes of blots containingSf-9S and Sf-9S infected cell proteins but not in lanes containingpurified recombinant proteins such as HPV-16 L1 proteins (FIG. 10B).

Similarly, antisera were produced in rabbits against antigens comprisedof wild-type baculovirus proteins expressed in Sf-9S and High Fiveinsect cells infected at an MOI of 3 pfu/cell with AcMNPV wild-typebaculovirus for three days to produce infected cell lysates. Theseantisera were utilized to demonstrate the presence of baculoviruscontaminants in baculovirus-derived recombinant protein products (FIG.10C). Little or no reactivity was observed in the lanes for recombinantHPV-16 L1 VLPs in Western blots with antisera to Sf-9S cell proteins orwild type baculovirus proteins, whereas polyclonal rabbit antisera toHPV-16 L1 proteins were positive for the recombinant HPV-16 L1 VLP lanes(FIG. 10A).

Example 22

Formulation of HPV-16 L1 VLP Monovalent Vaccine

Monovalent vaccines of BPV-16 VLPs were prepared by formulation ofrecombinant HPV-16 L1 VLPs manufactured by the methods in Example 18.Final bulk products of recombinant HPV-16 L1 VLPs were filtered througha 0.22 μm membrane aseptically and formulated at antigen concentrationsof 20 and 100 mg/ml in phosphate-buffered saline alone or with alumadjuvant (Alhydrogel). The formulated products were dispensed as singledose units (0.5 cc) into sterile borosilicate glass vials (3 cc)silanized with dimethyldichlorosilane.

Also, final bulk products of recombinant HPV-16 L1 VLPs were formulatedat antigen concentrations of 40 and 200 μg/ml in phosphate bufferedsaline, filtered, and dispensed as single dose units (0.25 cc) intosilanized glass vials (3 cc) for mixing just prior to immunization withan equal volume of the adjuvant MF-50 (Chiron). The final containerproducts were labeled, checked for vial integrity, and stored at −20 or−70° C. for final container vials containing VLP alone and 2-8° C. forfinal container vials containing VLPs and alum adjuvant.

The final container products were subjected to safety and analyticaltesting as required by United States federal regulations and passedproduct specifications. Safety specifications included: (1) the absenceof detectable microbial contaminants, spiroplasma, or mycoplasma, (2)endotoxins levels below 30 endotoxin units per ml, (3) the absence ofadventitious agents by in vitro and in vivo testing, and (4) no adverseeffects in adult mice and guinea pigs as part of the general safetytests. Analytical specifications included (1) the presence of HPV-16 LUproteins with molecular weights between 50 and 60 kD at a purity >95% asdetermined by SDS-PAGE analysis coupled to scanning laser densitometry,(2) for identity testing, positive reactivity of proteins in the productto HPV-16 L1 antisera as determined by Western blot analysis, (3) forpotency testing, positive reactivity of product at 100 μg/ml dilutionwith H16.V5 antisera for conformational epitopes as determined by ELISAtesting, (4) for identity, purity, and potency testing, at least 75% ofthe product was present as intact VLPs as determined by analytical sizeexclusion chromatography and negative-stain electron rmicroscopy, andfor strength testing, protein content in the product at 20 and 100 g/mlwith <5% variance as determined by BCA assay.

Upon meeting product specifications, the final container vials werereleased for vaccination into healthy human volunteers to determine thesafety and immunogenicity of the final container products asprophylactic vaccines for HPV-16 infection and disease.

Example 23

Clinical Investigation of HPV-16 VLP Monovalent Vaccine

The final container products as prepared by the methods of Example 22were used to immunize healthy human volunteers in a double-blind,randomized clinical study at Johns Hopkins University to determine thesafety and immunogenicity of the present invention as a monovalentvaccine to prevent HPV-16 infection and disease (Harro et al., 2001).The study design encompassed two dosage regimens (10 and 50 μg VLPantigens) and four study arms including placebo, HPV-16 L1 VLPs alone,HPV-16 L1 VLPs +alum adjuvant, and HPV-16 L1 VLPs+MF-59 adjuvant. Femalevolunteers (72) who were sero-negative for HIV-1, had <four lifetimesexual partners, were not pregnant, and had normal cervical cytology andmedical history, received three doses of vaccine or placebointramuscularly in the deltoid muscle area at 0, 1, and 4 months. Serumsamples were collected from vaccinees at one month post-immunization andevaluated by ELISA tests for the presence of HPV-16 antibodies.Vaccinees were followed up to one week post-immunization forpresentation of adverse clinical signs.

The results of the clinical study indicated that the vaccine waswell-tolerated as compared to placebo, as no major adverse effects werenoted in any vaccinees. All vaccines receiving active vaccineseroconverted (4-fold antibody rise) for HPV-16. A dose-dependent immuneresponse was observed in serum samples at 5 months post-immunization forthose vaccinees receiving VLPs alone or VLPs+Mf-59. However, no dosedependent response was seen in individuals receiving VLPs+alum. Theantibody titers to HPV-16 neutralizing epitopes, as determined by ELISAtests of serum samples at five months post-immunization with 50 μgdoses, were 1×10⁴ E.U. for VLPs alone, 1×10⁴ E.U. for VLPs+MF-59, and2.2×10³ E.U. for VLPs+alum. The neutralizing and ELISA antibody titerswere shown to correlate well with 0.85 degree of confidence. Thus, thevaccines were shown to be well-tolerated and immunogenic. The antibodytiters were approximately forty-fold higher than that associated withnatural HPV-16 infections. ELISA antibody titers were demonstrated to bereliable correlates of HPV-16 neutralizing antibody titers. Lastly,HPV-16 L1 VLP vaccines consisting of VLPs alone at 50 μg per dose mayprovide protective immunity to prevent HPV-16 infection and resolveactive HPV-16 infections and disease.

All references discussed herein are incorporated by reference. Oneskilled in the art will readily appreciate that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The present invention maybe embodied in other specific forms without departing from the spirit oressential attributes thereof and, accordingly, reference should be madeto the appended claims, rather than to the foregoing specification, asindicating the scope of the invention.

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1. A chimeric virus-like particle comprising a recombinant viral capsidprotein that encapsulates a recombinant viral protein during selfassembly into a chimeric virus-like particle, wherein the chimericvirus-like particle exhibits conformational antigenic epitopes capableof eliciting neutralizing antibodies.
 2. The chimeric virus-likeparticle of claim 1, wherein the recombinant viral capsid protein andthe recombinant viral protein are from the same or a different virus. 3.The chimeric virus-like particle of claim 1, wherein the recombinantviral capsid protein or the recombinant viral protein is from anenveloped virus, a non-enveloped virus, or both.
 4. The chimericvirus-like particle of claim 3, wherein the enveloped virus comprisesinfluenza virus, hepatitis C virus, and human immunodeficiency virus. 5.The chimeric virus-like particle of claim 3, wherein the non-envelopedvirus comprises rotavirus, calicivirus, hepatitis E virus,papillomavirus, influenza virus, and hepatitis C viris.
 6. The chimericvirus-like particle of claim 5, wherein the papillomavirus compriseshuman papillomavirus.
 6. The chimeric virus-like particle of claim 5,wherein the viral capsid protein and the viral protein are from the sameor a different human papillomavirus genotype.
 7. The chimeric virus-likeparticle of claim 6, wherein the viral capsid protein comprises a HPV L1protein and the viral protein comprises a HPV L2, or a HPV L2 fusionprotein.
 8. The chimeric virus-like particle of claim 7, wherein the HPVL2 fusion protein comprises HPV L2, fused with HPV E2, HPV E6, and/orHPV E7.
 9. The chimeric virus-like particle of claim 5 wherein the humanpapillomavirus genotypes comprise HPV-16, HPV-18, HPV-45, HPV-31,HPV-33, HP]V-35, HPV-51, HPV-52, HPV-6, HPV-11, HPV-42, HPV-43, HPV-44,or a combination thereof.
 10. The chimeric virus-like particle of claim1, wherein the viral capsid protein is encoded by a codon optimizedpolynucleotide comprising SEQ ID No. 1, or a polynucleotide having asequence that is substantially homologous to SEQ ID No.
 1. 11. Thechimeric virus-like particles of claim 1, wherein the codon optimizedpolynucleotide encoding the viral protein comprises SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No. 4, SEQ ID No. 5, or a polynucleotide having a sequencethat is substantially homologous to SEQ ID No.2, SEQ ID No. 3, SEQ IDNo. 4, or SEQ ID No.
 5. 12. The chimeric virus-like particle of claim 8,wherein the fusion protein comprises a heterologous protein.
 13. Amethod for producing chimeric virus-like particles comprising: (a)infecting an insect cell with a first recombinant baculovirus carrying afirst codon-optimized polynucleotide that encodes a first viral protein;(b) infecting the insect cell of the step (a) with a second recombinantbaculovirus carrying a second codon-optimized polynucleotide thatencodes a second viral protein, wherein the first viral proteincomprises a recombinant viral capsid protein that encapsulates thesecond viral protein during assembly of virus-like particles.
 14. Themethod of claim 13, wherein the first recombinant baculovirus and thesecond recombinant baculovirus are co-infected into the insect cell. 15.The method of claim 13, wherein the insect cell is from the cell linedesignated ATCC PTA4047.
 16. The method of claim 13, wherein therecombinant viral capsid protein comprises a HPV L1 protein and thesecond viral protein is a recombinant viral protein comprising a HPV L2or a HPV L2 fusion protein.
 17. The method of claim 14, whereinco-infection of the first and second recombinant baculoviruses resultsin an expression ratio of at least about 3:1 for the second viralprotein versus the recombinant viral capsid protein.
 18. A codonoptimized polynucleotide comprising at least one of the followingcharacteristics: (a) an increased number of nucleotide sequences thatare utilized at high levels in insect cells, (b) a ratio of GCnucleotide pairs to AT nucleotide pairs of approximately 1: 1, (c) aminimum number of palindromic and stem-loop DNA structures, and (d) aminimum number of transcription and post-transcription repressorelements.
 19. The codon optimized polynucleotide of claim 18 encoding aviral capsid protein, or encoding a viral protein that becomesencapsulated into a virus like particle by the viral capsid protein inan insect cell.
 20. The codon optimized polynucleotide of claim 19comprising a polynucleotide encoding a protein from an enveloped virus,a non-enveloped virus, or both.
 21. The codon optimized polynucleotideof claim 20, wherein the enveloped virus comprises influenza virus,hepatitis C virus, and human immunodeficiency virus.
 22. The codonoptimized polynucleotide of claim 20, wherein the non-enveloped viruscomprises rotavirus, calicivirus, hepatitis E virus, papillomavirus,influenza virus, and hepatitis C virus.
 23. The codon optimizedpolynucleotide of claim 20, wherein the protein is HPV L2 protein or apeptide fragment thereof.
 24. The codon optimized polynucleotide ofclaim 20, wherein the protein is a HPV L2 fusion protein comprisingheterologous proteins.
 25. The codon optimized polynucleotide of claim18 represented by SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No.5, or a sequence that is substantially homologous to SEQ ID No.2, SEQ IDNo. 3, SEQ ID No. 4, or SEQ ID No.
 5. 26. A vector comprising the codonoptimized polynucleotide of claim 18 operatively linked to a baculovirusregulatory control element, which vector is capable of replication in aninsect host cell.
 27. An insect host cell comprising the vector of claim26.
 28. A pharmaceutical composition for treating or preventing apapillomavirus related disease or disorder comprising administering to asubject in need thereof a composition containing chimeric virus-likeparticles that exhibit conformational antigenic epitopes capable ofeliciting neutralizing antibodies in the subject, wherein the chimericvirus-like particles comprise a recombinant HPV L1 that encapsulates arecombinant TPV L2 and/or HPV L2 fusion protein, and an acceptablecarrier or diluent.
 29. The pharmaceutical composition of claim 28,where the HPV L1, HPV L2, and/or HPV L2 fusion proteins are from thesame or a different HPV genotypes.
 30. A vaccine composition forinducing immunity against a papillomavirus infection in a humancomprising a composition containing chimeric virus-like particles thatexhibit conformational antigenic epitopes capable of elicitingneutralizing antibodies in the human, wherein the chimeric virus-likeparticles comprise a recombinant HPV L1 that encapsulates a recombinantHPV L2 and/or HPV L2 fusion protein, and an adjuvant.
 31. The vaccinecomposition of claim 30, wherein the immunity is humoral immunity, cellmediated immunity, or both.
 32. The vaccine composition of claim 30,wherein the infecting papillomavirus comprises a human papillomavirusgenotype selected from the group consisting of BPV-16, HPV-18, BPV-45,HPV-31, HPV-33, HPV-35, HPV-51, HPV-52, HPV-6, HPV-11, HPV42, HPV-43,HPV-44, and combinations thereof.
 32. The vaccine composition of claim30 comprising a monovalent formulation.
 33. The vaccine composition ofclaim 30 comprising a multivalent formulation.
 34. The vaccinecomposition of claim 30, comprising a) a polypeptide which is encoded bya polynucleotide molecule represented by SEQ ID No. 2, SEQ ID No. 3, SEQID No. 4, SEQ ID No. 5, or a polynucleotide molecule that issubstantially homologous to SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, orSEQ ID No. 5; b) a polynucleotide molecule represented by SEQ ID No. 2,SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or a polynucleotide moleculethat is substantially homologous to SEQ ID No.2, SEQ ID No. 3, SEQ IDNo. 4, or SEQ ID No. 5; or c) a vector carrying a polynucloetidemolecule represented by SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ IDNo. 5, or a polynucleotide molecule that is substantially homologous toSEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5; and anadjuvant.
 35. A diagnostic test kit for detection of papillomavirusinfection, comprising a composition containing chimeric virus-likeparticles that exhibit conformational antigenic epitopes capable ofeliciting neutralizing antibodies in a subject, wherein the chimericvirus-like particles comprise a recombinant HPV L1 that encapsulates arecombinant HPV L2 and/or HPV L2 fusion protein, and a detection agent.36. The diagnostic test kit of claim 34, wherein the papillomavirusinfection is caused by one or more human papillomavirus genotypes.
 37. Amethod of treating or preventing a papillomavirus related disease ordisorder comprising administering to an individual in need thereof aneffective amount of the pharmaceutical composition of claim
 28. 38. Amethod of protecting an individual against a papillomavirns infection,comprising administering to the individual an effective amount of thevaccine of claim 29.